RESUMEN
The present study tested the hypothesis that protein kinase C-α (PKC-α) recruitment in the presence of the p38α/ß MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day-old NNVMs but significantly increased runt-related transcription factor 1 (Runx1) and p16 cell cycle inhibitor (CDKN2a) mRNA levels and downregulated epithelial cell transforming 2 (ECT2) mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu, 1.4 ± 3 vs. PDBu/SB203580, 128 ± 34; n = 5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-α, protein kinase-δ (PKC-δ) and protein kinase-ε (PKC-ε) protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-α protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 40 ± 46; n = 5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 192 ± 42; n = 5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(+)-NNVMs (PDBu/SB203580/sotrastaurin, 8 ± 10; n = 4) and nestin(-)-NNVMs (PDBu/SB203580/sotrastaurin, 64 ± 30; n = 4) that incorporated 5-bromo-2'-deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.NEW & NOTEWORTHY The appearance of nestin(+)-neonatal rat ventricular cardiomyocytes that re-entered the cell cycle following phorbol ester stimulation in the presence of p38α/ß MAPK inhibitor SB203580 was associated with the inhibition of Runx1 and CDKN2a mRNA upregulation. PKC-α selectively induced the cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes. Pharmacological inhibition of PKC-α with concomitant p38α/ß MAPK suppression unmasked the cell cycle re-entry of a second population of neonatal rat ventricular cardiomyocytes in the absence of nestin expression.
Asunto(s)
Miocitos Cardíacos , Proteína Quinasa C , Ratas , Animales , Proteína Quinasa C/metabolismo , Miocitos Cardíacos/metabolismo , Animales Recién Nacidos , Nestina/genética , Nestina/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Bromodesoxiuridina , Ciclo Celular , Isoformas de Proteínas , ARN Mensajero/genética , Forbol 12,13-Dibutirato/farmacologíaRESUMEN
Protein kinase C (PKC) activation can evoke vasoconstriction and contribute to coronary disease. However, it is unclear whether PKC activation, without activating the contractile machinery, can lead to coronary arteriolar dysfunction. The vasoconstriction induced by the PKC activator phorbol 12,13-dibutyrate (PDBu) was examined in isolated porcine coronary arterioles. The PDBu-evoked vasoconstriction was sensitive to a broad-spectrum PKC inhibitor but not affected by inhibiting PKCß2 or Rho kinase. After exposure of the vessels to a sub-vasomotor concentration of PDBu (1 nmol/L, 60 min), the endothelium-dependent nitric oxide (NO)-mediated dilations in response to serotonin and adenosine were compromised but the dilation induced by the NO donor sodium nitroprusside was unaltered. PDBu elevated superoxide production, which was blocked by the superoxide scavenger Tempol. The impaired NO-mediated vasodilations were reversed by Tempol or inhibition of PKCß2, xanthine oxidase, c-Jun N-terminal kinase (JNK) and Rho kinase but were not affected by a hydrogen peroxide scavenger or inhibitors of NAD(P)H oxidase and p38 kinase. The PKCß2 protein was detected in the arteriolar wall and co-localized with endothelial NO synthase. In conclusion, activation of PKCß2 appears to compromise NO-mediated vasodilation via Rho kinase-mediated JNK signaling and superoxide production from xanthine oxidase, independent of the activation of the smooth muscle contractile machinery.
Asunto(s)
Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , Óxido Nítrico/metabolismo , Proteína Quinasa C beta/metabolismo , Vasodilatación , Animales , Inmunohistoquímica , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C beta/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Porcinos , Vasodilatación/genética , Vasodilatadores/farmacología , Xantina Oxidasa/metabolismoRESUMEN
Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/ß mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.
Asunto(s)
Corazón/crecimiento & desarrollo , Proteína Quinasa 14 Activada por Mitógenos/genética , Nestina/biosíntesis , Miosina Tipo IIB no Muscular/genética , Organogénesis/genética , Animales , Animales Recién Nacidos/genética , Animales Recién Nacidos/crecimiento & desarrollo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/genética , Regulación del Desarrollo de la Expresión Génica/genética , Corazón/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/crecimiento & desarrollo , Humanos , Imidazoles/farmacología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Nestina/genética , Forbol 12,13-Dibutirato/farmacología , Piridinas/farmacología , Ratas , Pez Cebra/genéticaRESUMEN
BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.
Asunto(s)
Membrana Celular/enzimología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteína Quinasa C-alfa/metabolismo , Vasodilatación , Proteínas de Unión al GTP rho/metabolismo , Animales , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Forbol 12,13-Dibutirato/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Quinasas Asociadas a rho/metabolismoRESUMEN
The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.
Asunto(s)
Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/metabolismo , Aorta Torácica/fisiología , Contracción Isométrica/efectos de los fármacos , Músculo Liso Vascular/fisiología , Vasodilatadores/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Monoterpenos Ciclohexánicos , Ciclohexenos/química , Ciclohexenos/farmacología , Limoneno , Estructura Molecular , Monoterpenos/química , Monoterpenos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Fenilefrina/efectos adversos , Forbol 12,13-Dibutirato/efectos adversos , Ratas , Relación Estructura-Actividad , Terpenos/química , Terpenos/farmacología , Vasodilatadores/químicaRESUMEN
Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ⫠W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.
Asunto(s)
Proteínas del Tejido Nervioso/química , Triptófano/química , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Ligandos , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Forbol 12,13-Dibutirato/farmacología , Unión Proteica , Conformación Proteica/efectos de los fármacos , Dominios Proteicos , Transporte de Proteínas/efectos de los fármacos , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Increasing evidence suggests a functional role of RhoA/Rho-kinase signalling as a mechanism for smooth muscle contraction; however, little is known regarding the roles of Rac1 and other members of the Rho protein family. This study aimed to examine whether Rac1 modulates bronchial smooth muscle contraction. Ring preparations of bronchi isolated from rats were suspended in an organ bath, and isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine myosin light chain phosphorylation in bronchial smooth muscle. Our results demonstrated that muscle contractions induced by carbachol (CCh) and endothelin-1 (ET-1) were inhibited by EHT1864, a selective Rac1 inhibitor, and NSC23766, a selective inhibitor of Rac1-specific guanine nucleotide exchange factors. Similarly, myosin light chain and myosin phosphatase target subunit 1 (MYPT1) at Thr853 phosphorylation induced by contractile agonist were inhibited with Rac1 inhibition. However, contractions induced by high K+, calyculin A (a potent protein phosphatase inhibitor) and K+/PDBu were not inhibited by these Rac1 inhibitors. Interestingly, NaF (a G-protein activator)-induced contractions were inhibited by EHT1864 but not by NSC23766. We next examined the effects of a trans-acting activator of transcription protein transduction domain (PTD) fusion protein with Rac1 (PTD-Rac1) on muscle contraction. The constitutively active form of PTD-Rac1 directly induced force development and contractions were abolished by EHT1864. These results suggest that Rac1, activated by G protein-coupled receptor agonists, such as CCh and ET-1, may induce myosin light chain and MYPT phosphorylation and modulate the contraction of bronchial smooth muscle.
Asunto(s)
Bronquios/fisiología , Contracción Muscular , Músculo Liso/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Bronquios/efectos de los fármacos , Carbacol/farmacología , Endotelina-1/farmacología , Masculino , Toxinas Marinas , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Oxazoles/farmacología , Forbol 12,13-Dibutirato/farmacología , Fosforilación/efectos de los fármacos , Potasio/metabolismo , Proteína Fosfatasa 1/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: Although physiologic transition from rhythmic contractions to uterine retraction postpartum remains a poorly understood process, it has been shown that the latter is essential in the prevention of hemorrhage and its negative consequences. OBJECTIVE: To investigate the transition from oscillatory contractions to tonic contracture in human myometrium after delivery, a mechanism purported to facilitate postpartum hemostasis. Protein kinase C (PKC) plays a key regulatory role in human uterine contractions because it can prevent dephosphorylation of regulatory proteins and sensitize the contractile machinery to low Ca2+. Thus, activation of PKC by phorbol 12,13-dibutyrate (PDBu) may act as a strong uterotonic agent. STUDY DESIGN: Uterine biopsies were obtained from consenting women undergoing elective caesarian delivery at term without labor (N = 19). Isometric tension measurements were performed on uterine strips (n = 114). The amplitudes and area under the curve of phasic contractions and tonic responses were measured and compared. A total of 1 µM PDBu was added to the isolated organ baths, and maximal tension of the uterine contracture was determined in the absence and presence of either 1 µM of staurosporine, 100 nM nifedipine, or 10 µM cyclopiazonic acid to assess the role of PKC and calcium sensitivity on uterine contractility. RESULTS: On the addition of PDBu on either basal or oxytocin-induced activity, consistent contractures were obtained concomitant with complete inhibition of phasic contractions. After a 30-minute incubation period, the mean amplitude of the PDBu-induced tone represented 65.3% of the amplitude of spontaneous contraction. Staurosporine, a protein kinase inhibitor, induced a 91.9% inhibition of PDBu contractures, a process not affected by nifedipine or cyclopiazonic acid, thus indicating that this mechanism is largely Ca2+ independent. CONCLUSION: Pharmacologic activation of PKC leads to a significant contracture of the myometrium. Together, these data suggest that the up-regulation of PKC plays a physiologic role in the modulation of uterine contracture after delivery. A switch from phasic to strong tonic contractions potentially may facilitate postpartum hemostasis.
Asunto(s)
Miometrio/efectos de los fármacos , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/efectos de los fármacos , Contracción Uterina/efectos de los fármacos , Adulto , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Técnicas In Vitro , Indoles/farmacología , Miometrio/metabolismo , Nifedipino/farmacología , Proteína Quinasa C/metabolismo , Estaurosporina/farmacología , Tocolíticos/farmacología , Adulto JovenRESUMEN
Mechanisms underlying the vasorelaxant effects of trans-4-methyl-ß-nitrostyrene (T4MeN) were studied in rat aortic rings. In endothelium-intact preparations, T4MeN fully and similarly relaxed contractions induced by phenylephrine (PHE) (IC50 = 61.41 [35.40-87.42] µmol/L) and KCl (IC50 = 83.50 [56.63-110.50] µmol/L). The vasorelaxant effect of T4MeN was unchanged by endothelium removal, pretreatment with L-NAME, indomethacin, tetraethylammonium, ODQ or MDL-12,330A. Under Ca2+ -free conditions, T4MeN significantly reduced with a similar potency: (i) phasic contractions induced by PHE, but not by caffeine; (ii) contractions due to CaCl2 in aortic preparations stimulated with PHE (in the presence of verapamil) or high KCl; (iii) contractions evoked by the restoration of external Ca2+ levels after depletion of intracellular Ca2+ stores in the presence of thapsigargin. In contrast, T4MeN was more potent at inhibiting contractions evoked by the tyrosine phosphatase inhibitor, sodium orthovanadate, than those induced by the activator of PKC, phorbol-12,13-dibutyrate. These results suggest that T4MeN induces an endothelium- independent vasorelaxation that appears to occur intracellularly through the inhibition of contractions that are independent of Ca2+ influx from the extracellular milieu but involve phosphorylation of tyrosine residues.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Estirenos/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/síntesis química , Vasodilatadores/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Técnicas de Química Sintética , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Forbol 12,13-Dibutirato/farmacología , Cloruro de Potasio/farmacología , Ratas , Estirenos/química , Vanadatos/farmacología , Vasodilatadores/químicaRESUMEN
Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 µg/mL-azithromycin and 10 and 50 µg/mL-chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.
Asunto(s)
Azitromicina/administración & dosificación , Cloranfenicol/administración & dosificación , Trampas Extracelulares/efectos de los fármacos , Infecciones/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Trampas Extracelulares/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Infecciones/inmunología , Infecciones/patología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Forbol 12,13-Dibutirato/químicaRESUMEN
Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Miocitos Cardíacos/metabolismo , Proteína Quinasa C/metabolismo , Antracenos/farmacología , Cloruros/agonistas , Cloruros/antagonistas & inhibidores , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Atrios Cardíacos/citología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Humanos , Soluciones Hipotónicas/metabolismo , Soluciones Hipotónicas/farmacología , Indoles/farmacología , Transporte Iónico/efectos de los fármacos , Maleimidas/farmacología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Forbol 12,13-Dibutirato/farmacología , Cultivo Primario de CélulasRESUMEN
PURPOSE: Resveratrol is a red wine polyphenol that causes vasorelaxation, which could be of interest in the treatment or prevention of eye diseases with an impaired blood flow. In this study, the vasorelaxant capacity of resveratrol (cis and trans) on bovine retinal arteries, its vasorelaxing mechanism, and its influence on the relaxation induced by the retinal relaxing factor (RRF) were examined. METHODS: Isolated bovine retinal arteries were mounted into wire myographs for isometric tension measurements. Concentration-response curves of cis- and trans-resveratrol and concentration-response curves of resveratrol in the absence or presence of the endothelium or different inhibitors were constructed. Relaxations elicited by the RRF with and without resveratrol incubation were also compared. RESULTS: Both resveratrol isomers caused a similar strong concentration-dependent relaxation. Removal of the endothelium or blocking endothelium-dependent pathways did not change the relaxation. Also, K+ channel blockers did not reduce the relaxation, except the 120 mM K+ Krebs Ringer bicarbonate solution. Phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate blocked the relaxation partially and so did the inhibition of heme oxygenase-1. Blocking adenylyl cyclase, AMP-activated protein kinase, estrogen receptors, sirtuin 1, or sarco/endoplasmic reticulum Ca2+ ATPase did not have an effect. The relaxation caused by the RRF was not altered by resveratrol incubation. CONCLUSIONS: Cis- and trans-resveratrol relax bovine retinal arteries similarly and concentration dependently. The main relaxation mechanism remains unclear, but K+ channels, carbon monoxide, and the myosin phosphatase pathway may be involved. Resveratrol does not have an influence on the RRF.
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Antioxidantes/farmacología , Arteria Retiniana/fisiología , Estilbenos/farmacología , Vasodilatación/fisiología , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Contracción Isométrica , Músculo Liso Vascular/efectos de los fármacos , Miografía , Óxido Nítrico/fisiología , Forbol 12,13-Dibutirato/farmacología , Canales de Potasio/fisiología , Proteína Quinasa C/fisiología , Resveratrol , Acetato de Tetradecanoilforbol/farmacología , Vasodilatadores/farmacologíaRESUMEN
The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.
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Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Forbol 12,13-Dibutirato/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
SCOPE: High-salt diet (HSD) is associated with cardiovascular diseases. This study aims at ascertaining the influence of maternal HSD on offspring's angiotensin II (ANG II)-mediated vasoconstriction and the underlying mechanisms. METHODS AND RESULTS: In comparison to a normal-salt diet, HSD used in pregnancy in rats changed the ultrastructures of the coronary artery (CA) in 5-month-old male offspring, and increased ANG II-mediated CA contractility. Measurement of [Ca(2+) ]i in CA using fluorescent fura-2, a Ca(2+) indicator, showed that ANG II-mediated increases in [Ca(2+) ]i were the same between HSD and normal-salt diet groups, but the ratio of diameter change/[Ca(2+) ]i induced by ANG II were significantly higher in HSD groups. Angiotensin II receptor type 1, not angiotensin II receptor type 2, caused ANG II-mediated vasoconstriction. Protein kinase C (PKC) inhibitor GF109203X attenuated the ANG II-mediated vasoconstriction, PKC agonist phorbol12,13-dibutyrate produced a greater contraction. There was an increase in PKCß mRNA and the corresponding protein abundance in the offspring, whereas other PKC subunits PKCα, PKCδ, and PKCε did not change. Moreover, 20 kDa myosin light chain phosphorylation levels were increased in HSD group. CONCLUSION: Maternal HSD affected the developmental programing for the offspring CA, with increased ANG II-mediated vasoconstrictions. The angiotensin II receptor type 1-PKC-20 kDa myosin light chain phosphorylation pathway was the possible mediated cellular mechanism.
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Fenómenos Fisiologicos Nutricionales Maternos , Efectos Tardíos de la Exposición Prenatal/genética , Receptor de Angiotensina Tipo 1/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Vasoconstricción/efectos de los fármacos , Angiotensina II/sangre , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Dieta , Femenino , Indoles/farmacología , Masculino , Maleimidas/farmacología , Cadenas Ligeras de Miosina/metabolismo , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Embarazo , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/genética , Renina/sangre , Sistema Renina-Angiotensina/efectos de los fármacos , Transducción de Señal , Sodio/sangreRESUMEN
Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Etanol/farmacología , Neuronas/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores de GABA-A/efectos de los fármacos , Animales , Bicuculina/farmacología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Antagonistas del GABA/farmacología , Isoxazoles/farmacología , Masculino , Forbol 12,13-Dibutirato/farmacología , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/enzimologíaRESUMEN
BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²⁺]ᵢ) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²⁺]ᵢ. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.
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Animales , Ratas , Anestésicos Locales , Aorta , Western Blotting , Bupivacaína , Calcio , Fura-2 , Técnicas In Vitro , Mepivacaína , Músculo Liso Vascular , Fosfatasa de Miosina de Cadena Ligera , Forbol 12,13-Dibutirato , Fosforilación , Proteína Quinasa C , Solubilidad , VasodilataciónRESUMEN
BACKGROUND: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. METHODS: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²⁺]ᵢ) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. RESULTS: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²⁺]ᵢ. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. CONCLUSIONS: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.
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Animales , Ratas , Anestésicos Locales , Aorta , Western Blotting , Bupivacaína , Calcio , Fura-2 , Técnicas In Vitro , Mepivacaína , Músculo Liso Vascular , Fosfatasa de Miosina de Cadena Ligera , Forbol 12,13-Dibutirato , Fosforilación , Proteína Quinasa C , Solubilidad , VasodilataciónRESUMEN
Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
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Humanos , Antracenos , Farmacología , Canales de Cloruro , Metabolismo , Cloruros , Metabolismo , Medios de Cultivo , Metabolismo , Farmacología , Relación Dosis-Respuesta a Droga , Potenciales Evocados , Fisiología , Atrios Cardíacos , Biología Celular , Metabolismo , Soluciones Hipotónicas , Metabolismo , Farmacología , Indoles , Farmacología , Transporte Iónico , Maleimidas , Farmacología , Miocitos Cardíacos , Biología Celular , Metabolismo , Técnicas de Placa-Clamp , Forbol 12,13-Dibutirato , Farmacología , Cultivo Primario de Células , Proteína Quinasa C , MetabolismoRESUMEN
Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABAB pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism. PKC phosphorylation acts on the channel to reduce activity, yet the mechanism is incompletely understood. Using the heterologous Xenopus oocyte system combined with molecular dynamics simulations, we show that PKC modulation of channel activity is dependent on Ser-196 in Kir3.2 such that, when this site is phosphorylated, the channel is less sensitive to PKC inhibition. This reduced inhibition is dependent on an interaction between phospho-Ser (SEP)-196 and Arg-201, reducing Arg-201 interaction with the sodium-binding site Asp-228. Neutralization of either SEP-196 or Arg-201 leads to a channel with reduced activity and increased sensitivity to PKC inhibition. This study clarifies the role of Ser-196 as an allosteric modulator of PKC inhibition and suggests that the SEP-196/Arg-201 interaction is critical for maintaining maximal channel activity. SIGNIFICANCE STATEMENT: The inwardly rectifying potassium 3.2 (Kir3.2) channel is found principally in neurons that regulate diverse brain functions, including pain perception, alcoholism, and substance addiction. Activation or inhibition of this channel leads to changes in neuronal firing and chemical message transmission. The Kir3.2 channel is subject to regulation by intracellular signals including sodium, G-proteins, ethanol, the phospholipid phosphatidylinositol bis-phosphate, and phosphorylation by protein kinases. Here, we take advantage of the recently published structure of Kir3.2 to provide an in-depth molecular view of how phosphorylation of a specific residue previously thought to be the target of PKC promotes channel gating and acts as an allosteric modulator of PKC-mediated inhibition.
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Fenómenos Biofísicos/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Activación del Canal Iónico/genética , Potenciales de la Membrana/fisiología , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Microinyecciones , Modelos Moleculares , Oocitos , Técnicas de Placa-Clamp , Forbol 12,13-Dibutirato/farmacología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilación , Mutación Puntual/genética , Proteína Quinasa C/metabolismo , Serina/genética , Xenopus laevisRESUMEN
The expression of an α-subunit of interleukin-2 receptor (IL-2Rα) was assessed by quantifying activation-induced upregulation of CD25 in IL-2-independent Jurkat leukemic cell line. It has been found that in growing Jurkat culture within 24 h, phytohemagglutinin (PHA, 5 µg/ml) or PHA in combination with 12,13-phorbol dibutirate (PDBu, 10(-8)M) increase the number of CD25+ cells to 32.3 ± 3.4% (n = 11) and 44.8 ± 8.6% (n = 6) respectively. Interleukin-2 (IL-2, 200 U/ml) alone or in combination with PDBu did not induce CD25 expression in Jurkat cells. All the tested stimulatory agencies affected neither the proliferation status no the growth of Jurkat cell cultures. In contrast to human blood T cells, WHI-P131, a selective pharmacological inhibitor of JAK/STAT signaling and CD25 expression, did not decrease the number of induced CD25+ cells in Jurkat culture. Flow cytometry analysis revealed a dose-dependent decrease in the proportion of cells in G1 phase and an increase in the proportion of cells in G2/M phase in WHI-P131-treated Jurkat cultures. It has been also found that WHI-P131 induces G2/M arrest in the absence of PHA or PDBu. We have concluded that (1) the IL-2-independent T cells of Jurkat line had not loss the mechanism for IL-2Rα expression in response to T cell receptor activation, (2) in the transformed T cells, WHI-P131 can arrest cell cycle at G2/M phase and has effects on targets other than IL-2 receptor-associated tyrosine kinase JAK3.