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1.
Biochemistry (Mosc) ; 88(8): 1126-1138, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37758312

RESUMO

Hyperglycemia is a hallmark of type 2 diabetes implicated in vascular endothelial dysfunction and cardiovascular complications. Many in vitro studies identified endothelial apoptosis as an early outcome of experimentally modeled hyperglycemia emphasizing cell demise as a significant factor of vascular injury. However, endothelial apoptosis has not been observed in vivo until the late stages of type 2 diabetes. Here, we studied the long-term (up to 4 weeks) effects of high glucose (HG, 30 mM) on human umbilical vein endothelial cells (HUVEC) in vitro. HG did not alter HUVEC monolayer morphology, ROS levels, NO production, and exerted minor effects on the HUVEC apoptosis markers. The barrier responses to various clues were indistinguishable from those by cells cultured in physiological glucose (5 mM). Tackling the key regulators of cytoskeletal contractility and endothelial barrier revealed no differences in the histamine-induced intracellular Ca2+ responses, nor in phosphorylation of myosin regulatory light chain or myosin light chain phosphatase. Altogether, these findings suggest that vascular endothelial cells may well tolerate HG for relatively long exposures and warrant further studies to explore mechanisms involved in vascular damage in advanced type 2 diabetes.

2.
Int J Mol Sci ; 23(1)2021 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-35008640

RESUMO

Hyperlipidemia manifested by high blood levels of free fatty acids (FFA) and lipoprotein triglycerides is critical for the progression of type 2 diabetes (T2D) and its cardiovascular complications via vascular endothelial dysfunction. However, attempts to assess high FFA effects in endothelial culture often result in early cell apoptosis that poorly recapitulates a much slower pace of vascular deterioration in vivo and does not provide for the longer-term studies of endothelial lipotoxicity in vitro. Here, we report that palmitate (PA), a typical FFA, does not impair, by itself, endothelial barrier and insulin signaling in human umbilical vein endothelial cells (HUVEC), but increases NO release, reactive oxygen species (ROS) generation, and protein labeling by malondialdehyde (MDA) hallmarking oxidative stress and increased lipid peroxidation. This PA-induced stress eventually resulted in the loss of cell viability coincident with loss of insulin signaling. Supplementation with 5-aminoimidazole-4-carboxamide-riboside (AICAR) increased endothelial AMP-activated protein kinase (AMPK) activity, supported insulin signaling, and prevented the PA-induced increases in NO, ROS, and MDA, thus allowing to maintain HUVEC viability and barrier, and providing the means to study the long-term effects of high FFA levels in endothelial cultures. An upgraded cell-based model reproduces FFA-induced insulin resistance by demonstrating decreased NO production by vascular endothelium.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Endotélio Vascular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Palmitatos/metabolismo , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
J Pept Sci ; 22(11-12): 673-681, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27699916

RESUMO

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Proteínas Aviárias/antagonistas & inibidores , Peptídeos Penetradores de Células/síntese química , Células Endoteliais/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/isolamento & purificação , Química Encefálica , Bovinos , Linhagem Celular , Peptídeos Penetradores de Células/sangue , Peptídeos Penetradores de Células/farmacologia , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Moela das Aves/química , Meia-Vida , Humanos , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/isolamento & purificação , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica , Proteólise , Técnicas de Síntese em Fase Sólida/métodos , Trombina/antagonistas & inibidores , Trombina/farmacologia , Perus
4.
Biomedicines ; 12(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38397940

RESUMO

Saturated free fatty acids are thought to play a critical role in metabolic disorders associated with obesity, insulin resistance, type 2 diabetes (T2D), and their vascular complications via effects on the vascular endothelium. The most abundant saturated free fatty acid, palmitate, exerts lipotoxic effects on the vascular endothelium, eventually leading to cell death. Shear stress activates the endothelial AMP-activated protein kinase (AMPK), a cellular energy sensor, and protects endothelial cells from lipotoxicity, however their relationship is uncertain. Here, we used isoform-specific shRNA-mediated silencing of AMPK to explore its involvement in the long-term protection of macrovascular human umbilical vein endothelial cells (HUVECs) against palmitate lipotoxicity and to relate it to the effects of shear stress. We demonstrated that it is the α1 catalytic subunit of AMPK that is critical for HUVEC protection under static conditions, whereas AMPK-α2 autocompensated a substantial loss of AMPK-α1, but failed to protect the cells from palmitate. Shear stress equally protected the wild type HUVECs and those lacking either α1, or α2, or both AMPK-α isoforms; however, the protective effect of AMPK reappeared after returning to static conditions. Moreover, in human adipose microvascular endothelial cells isolated from obese diabetic individuals, shear stress was a strong protector from palmitate lipotoxicity, thus highlighting the importance of circulation that is often obstructed in obesity/T2D. Altogether, these results indicate that AMPK is important for vascular endothelial cell protection against lipotoxicity in the static environment, however it may be dispensable for persistent and more effective protection exerted by shear stress.

5.
Biomedicines ; 10(12)2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36551937

RESUMO

Angiopathy is a common complication of diabetes mellitus. Vascular endothelium is among the first targets to experience blood-borne metabolic alterations, such as hyperglycemia and hyperlipidemia, the hallmarks of type 2 diabetes. To explore mechanisms of vascular dysfunction and eventual damage brought by these pathologic conditions and to find ways to protect vasculature in diabetic patients, various research approaches are used including in vitro endothelial cell-based models. We present an analysis of the data available from these models that identifies early endothelial cell apoptosis associated with oxidative stress as the major outcome of mimicking hyperglycemia and hyperlipidemia in vitro. However, the fate of endothelial cells observed in these studies does not closely follow it in vivo where massive endothelial damage occurs mainly in the terminal stages of diabetes and in conjunction with comorbidities. We propose that the discrepancy is likely in missing essentials that should be available to cultured endothelial cells to adjust the metabolic state and withstand the immediate apoptosis. We discuss the role of carnitine, creatine, and AMP-activated protein kinase (AMPK) in suiting the endothelial metabolism for long-term function in diabetic type milieu in vitro. Engagement of these essentials is anticipated to expand diabetes research options when using endothelial cell-based models.

6.
Biochem J ; 429(2): 291-302, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20459395

RESUMO

KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at pCa>8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after approximately 25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC.


Assuntos
Colo/fisiologia , Contração Muscular/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Cálcio/metabolismo , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Galinhas , Colo/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Primers do DNA/genética , Feminino , Cobaias , Humanos , Técnicas In Vitro , Masculino , Toxinas Marinhas , Microcistinas/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/farmacologia , Octoxinol , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia
7.
Biochim Biophys Acta Mol Cell Res ; 1868(11): 119104, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34302892

RESUMO

BACKGROUND: Myosin light chain kinase (MLCK) is a Ca2+-calmodulin-dependent enzyme dedicated to phosphorylate and activate myosin II to provide force for various motile processes. In smooth muscle cells and many other cells, small MLCK (S-MLCK) is a major isoform. S-MLCK is an actomyosin-binding protein firmly attached to contractile machinery in smooth muscle cells. Still, it can leave this location and contribute to other cellular processes. However, molecular mechanisms for switching the S-MLCK subcellular localization have not been described. METHODS: Site-directed mutagenesis and in vitro protein phosphorylation were used to study functional roles of discrete in-vivo phosphorylated residues within the S-MLCK actin-binding domain. In vitro co-sedimentation analysis was applied to study the interaction of recombinant S-MLCK actin-binding fragment with filamentous actin. Subcellular distribution of phosphomimicking S-MLCK mutants was studied by fluorescent microscopy and differential cell extraction. RESULTS: Phosphorylation of S-MLCK actin-binding domain at Ser25 and/or Thr56 by proline-directed protein kinases or phosphomimicking these posttranslational modifications alters S-MLCK binding to actin filaments both in vitro and in cells, and induces S-MLCK subcellular translocation with no effect on the enzyme catalytic properties. CONCLUSIONS: Phosphorylation of the amino terminal actin-binding domain of S-MLCK renders differential subcellular targeting of the enzyme and may, thereby, contribute to a variety of context-dependent responses of S-MLCK to cellular and tissue stimuli. GENERAL SIGNIFICANCE: S-MLCK physiological function can potentially be modulated via phosphorylation of its actin recognition domain, a regulation distinct from the catalytic and calmodulin regulatory domains.


Assuntos
Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fosforilação , Proteínas Quinases/metabolismo
8.
Biochimie ; 168: 83-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31668993

RESUMO

Myosin activation contributes to the contractile forces that induce disturbances in the vascular endothelial integrity and promote protein-rich edema of the underlying tissues. Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) have been reported to phosphorylate myosin regulatory light chains (RLC) for myosin activation. However, the relative contribution and roles of these kinases are debatable and not understood in very detail. In this study, using a combinational inhibitory analysis of MLCK, ROCK, and their antagonist, myosin light chain phosphatase (MLCP), we show that the MLCK-dependent RLC mono-(Ser19)phosphorylation (P-RLC) is sufficient to induce the FITC-dextran hyperpermeability in EA.hy926 endothelial cells (EC) in response to thrombin. However, MLCK relies on the ROCK assistance that attenuates MLCP activity. On the other hand, MLCK supplies P-RLC myosin as an intermediate substrate to ROCK thus adding to a faster accumulation of di-(Thr18/Ser19)phosphorylated RLC (PP-RLC) by the latter kinase. ROCK also produces P-RLC but is solely responsible for the thrombin-induced PP-RLC generation in EA.hy926 EC and other cell types. Still, as a direct myosin activator, ROCK contributes less to endothelial hyperpermeability than MLCK. Our findings are consistent with a concerted complementary mutual interplay between ROCK and MLCK to activate endothelial myosin and elicit the thrombin-mediated EC barrier dysfunction.


Assuntos
Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Quinase de Cadeia Leve de Miosina/fisiologia , Quinases Associadas a rho/fisiologia , Células Cultivadas , Humanos , Contração Muscular/fisiologia , Cadeias Leves de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Transdução de Sinais
9.
Sci Rep ; 9(1): 4872, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890744

RESUMO

Severe hypoxia leads to decline in cardiac contractility and induces arrhythmic events in part due to oxidative damage to cardiomyocyte proteins including ion transporters. This results in compromised handling of Ca2+ ions that trigger heart contractile machinery. Here, we demonstrate that thiol-containing compounds such as N-acetylcysteine (NAC), glutathione ethyl ester (et-GSH), oxidized tetraethylglutathione (tet-GSSG), oxidized glutathione (GSSG) and S-nitrosoglutathione (GSNO) are capable of reducing negative effects of hypoxia on isolated rat cardiomyocytes. Preincubation of cardiomyocytes with 0.1 mM GSNO, 0.5 mM et-GSH, GSSG, tet-GSSG or with 10 mM NAC allows cells 5-times longer tolerate the hypoxic conditions and elicit regular Ca2+ transients in response to electric pacing. The shape of Ca2+ transients generated in the presence of GSNO, et-GSH and NAC was similar to that observed in normoxic control cardiomyocytes. The leader compound, GSNO, accelerated by 34% the recovery of normal contractile function of isolated rat heart subjected to ischemia-reperfusion. GSNO increased glutathionylation of Na,K-ATPase alpha-2 subunit, the principal ion-transporter of cardiac myocyte sarcolemma, which prevents irreversible oxidation of Na,K-ATPase and regulates its function to support normal Ca2+ ion handling in hypoxic cardiomyocytes. Altogether, GSNO appears effective cardioprotector in hypoxic conditions worth further studies toward its cardiovascular application.


Assuntos
Arritmias Cardíacas/tratamento farmacológico , Hipóxia Celular/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Arritmias Cardíacas/patologia , Sinalização do Cálcio/efeitos dos fármacos , Estimulação Elétrica , Glutationa/análogos & derivados , Glutationa/farmacologia , Dissulfeto de Glutationa/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Contração Muscular/efeitos dos fármacos , Contração Miocárdica/fisiologia , Técnicas de Cultura de Órgãos , Oxigênio/metabolismo , Ratos , S-Nitrosoglutationa/farmacologia , Compostos de Sulfidrila/farmacologia
10.
Oxid Med Cell Longev ; 2017: 9456163, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28421129

RESUMO

Background. Nitric oxide can successfully compete with oxygen for sites of electron-transport chain in conditions of myocardial hypoxia. These features may prevent excessive oxidative stress occurring in cardiomyocytes during sudden hypoxia-reoxygenation. Aim. To study the action of the potent stable NO donor dinitrosyl iron complex with glutathione (Oxacom®) on the recovery of myocardial contractile function and Ca2+ transients in cardiomyocytes during hypoxia-reoxygenation. Results. The isolated rat hearts were subjected to 30 min hypoxia followed by 30 min reoxygenation. The presence of 30 nM Oxacom in hypoxic perfusate reduced myocardial contracture and improved recovery of left ventricular developed pressure partly due to elimination of cardiac arrhythmias. The same Oxacom concentration limited reactive oxygen species generation in hypoxic cardiomyocytes and increased the viability of isolated cardiomyocytes during hypoxia from 12 to 52% and after reoxygenation from 0 to 40%. Oxacom prevented hypoxia-induced elevation of diastolic Ca2+ level and eliminated Ca2+ transport alterations manifested by slow Ca2+ removal from the sarcoplasm and delay in cardiomyocyte relaxation. Conclusion. The potent stable NO donor preserved cardiomyocyte integrity and improved functional recovery at hypoxia-reoxygenation both in the isolated heart and in cardiomyocytes mainly due to preservation of Ca2+ transport. Oxacom demonstrates potential for cardioprotection during hypoxia-reoxygenation.


Assuntos
Coração/efeitos dos fármacos , Ferro/farmacologia , Miocárdio/metabolismo , Óxidos de Nitrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Cálcio/metabolismo , Hipóxia Celular , Células Cultivadas , Glutationa/metabolismo , Masculino , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo
11.
Oxid Med Cell Longev ; 2017: 1625130, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29098058

RESUMO

BACKGROUND: Malondialdehyde (MDA), glyoxal (GO), and methylglyoxal (MGO) levels increase in atherosclerosis and diabetes patients. Recent reports demonstrate that GO and MGO cause vascular endothelial barrier dysfunction whereas no evidence is available for MDA. METHODS: To compare the effects of MDA, GO, or MGO on endothelial permeability, we used human EA.hy926 endothelial cells as a standard model. To study cortical cytoplasm motility and cytoskeletal organization in endothelial cells, we utilized time-lapse microscopy and fluorescent microscopy. To compare dicarbonyl-modified protein band profiles in these cells, we applied Western blotting with antibodies against MDA- or MGO-labelled proteins. RESULTS: MDA (150-250 µM) irreversibly suppressed the endothelial cell barrier, reduced lamellipodial activity, and prevented intercellular contact formation. The motile deficiency of MDA-challenged cells was accompanied by alterations in microtubule and microfilament organization. These detrimental effects were not observed after GO or MGO (250 µM) administration regardless of confirmed modification of cellular proteins by MGO. CONCLUSIONS: Our comparative study demonstrates that MDA is more damaging to the endothelial barrier than GO or MGO. Considering that MDA endogenous levels exceed those of GO or MGO and tend to increase further during lipoperoxidation, it appears important to reduce oxidative stress and, in particular, MDA levels in order to prevent sustained vascular hyperpermeability in atherosclerosis and diabetes patients.


Assuntos
Aterosclerose/complicações , Diabetes Mellitus/sangue , Células Endoteliais/metabolismo , Complicações do Diabetes , Humanos , Permeabilidade
12.
J Cardiovasc Pharmacol ; 41(5): 788-94, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12717111

RESUMO

Immunochemical and ultrastructural studies of the rat heart after a single injection of doxorubicin (2.2 or 0.44 mg/kg) were performed. Ventricles were taken for the study 2 h and 3 weeks after injection. The light and electron microscopy and immunohistochemical determination of collagens of I, III, and IV types and fibronectin using specific antibodies were implied. Quantitive immunoblotting was used to analyze the expression levels of cytoskeletal and extracellular matrix proteins such as desmin, tubulin, vinculin, fibronectin, kinase-related protein (KRP or telokin), and smooth muscle/nonmuscle myosin light-chain kinase (MLCK). Doxorubicin (2.2 mg/kg) did not influence the relative volume and structure of collagen network but distinctly reduced the density of fibronectin distribution and decreased the content of tubulin, fibronectin, MLCK, and KRP. After 3 weeks, an increased density and extension of collagen network were observed, indicating the development of diffuse fibrosis whereas the content of tubulin and KRP increased above control level by 50 +/- 2.3% and 20 +/- 5.2%, correspondingly. Similar but less pronounced alterations were observed following the administration of 0.44 mg/kg doxorubicin. The content of MLCK after both doses consistently remained about 30% below its level in untreated animals. Isolated chick embryo cardiomyocytes subjected to doxorubicin responded by a 26% increase in KRP expression 4 days after whereas the level of tubulin expression remained unchanged. Thus, the damage of myocardium after a single injection of a therapeutic dose of doxorubicin was followed by an increased expression of selected cytoskeletal and extracellular matrix proteins, suggesting their involvement in cardiac reparation.


Assuntos
Proteínas do Citoesqueleto/biossíntese , Doxorrubicina/efeitos adversos , Proteínas da Matriz Extracelular/biossíntese , Ventrículos do Coração/patologia , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Células Cultivadas , Embrião de Galinha , Fibrose , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Immunoblotting , Imuno-Histoquímica , Cinesinas , Masculino , Microscopia Eletrônica , Proteínas Musculares/biossíntese , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Quinase de Cadeia Leve de Miosina/biossíntese , Fragmentos de Peptídeos , Peptídeos , Ratos , Ratos Wistar
13.
Mol Cell Biochem ; 252(1-2): 173-81, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14577591

RESUMO

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP--kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase--MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.


Assuntos
Agonistas Adrenérgicos beta/farmacologia , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Proteínas Musculares/metabolismo , Agonistas Adrenérgicos beta/administração & dosagem , Animais , Western Blotting , Isoproterenol/administração & dosagem , Masculino , Ratos , Ratos Wistar
14.
J Muscle Res Cell Motil ; 23(4): 341-51, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12630709

RESUMO

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919-3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg2+. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg(2+)-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Miosinas/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a Calmodulina/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas , Cinesinas , Magnésio/metabolismo , Magnésio/farmacologia , Microscopia Eletrônica , Modelos Biológicos , Contração Muscular/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/genética , Músculo Liso/efeitos dos fármacos , Músculo Liso/ultraestrutura , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/ultraestrutura , Miosinas/efeitos dos fármacos , Miosinas/ultraestrutura , Polímeros/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estrutura Terciária de Proteína/fisiologia , Transfecção
15.
Exp Cell Res ; 298(2): 407-17, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15265689

RESUMO

Recently discovered 210-kDa myosin light chain kinase (MLCK-210) is identical to 108-130 kDa MLCK, the principal regulator of the myosin II molecular motor, except for the presence of a unique amino terminal extension. Our in vitro experiments and transfected cell studies demonstrate that the N-terminal half of MLCK-210 unique tail domain has novel microfilament and microtubule binding activity. Consistent with this activity, the MLCK-210 domain codistributes with microfilaments and microtubules in cultured cells and with soluble tubulin in nocodazole-treated cells. This domain is capable of aggregating tubulin dimers in vitro, causing bundling and branching of microtubules induced by taxol. The N-terminal actin-binding region of MLCK-210 has lower affinity to actin (K(d) = 7.4 microM) than its central D(F/V)RXXL repeat-based actin-binding site and does not protect stress fibers from disassembly triggered by MLCK inhibition in transfected cells. Obtained results suggest that while being resident on microfilaments, MLCK-210 may interact with other cytoskeletal components through its N-terminal domain. Based on available evidence, we propose a model in which MLCK-210 could organize cell motility by simultaneous control of cytoskeleton architecture and actomyosin activation through the novel protein scaffold function of the unique tail domain and the classical MLCK catalytic function of the kinase domain.


Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Linhagem Celular , Chlorocebus aethiops , Citoesqueleto/ultraestrutura , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Peso Molecular , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão , Fibras de Estresse/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo
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