RESUMO
Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.
Assuntos
Fluoretos , Quinase de Cadeia Leve de Miosina , Animais , Humanos , Fosforilação , Células CACO-2 , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Fluoretos/metabolismo , Cálcio/metabolismo , Actinas/metabolismo , Junções Íntimas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Intestinal epithelial barrier function is closely associated with the development of many intestinal diseases. Heat-killed Lacticaseibacillus paracasei (HK-LP) has been shown to improve intestinal health and enhance immunity. However, the function of HK-LP in the intestinal barrier is still unclear. This study characterized the inflammatory effects of seven HK-LP (1 µg/mL) on the intestinal barrier using lipopolysaccharide (LPS) (100 µg/mL)-induced Caco-2 cells. In this study, HK-LP 6105, 6115, and 6235 were selected, and their effects on the modulation of inflammatory factors and tight junction protein expression (claudin-1, zona occludens-1, and occludin) were compared. The effect of different cultivation times (18 and 48 h) was investigated in response to LPS-induced intestinal epithelial barrier dysfunction. Our results showed that HK-LP 6105, 6115, and 6235 improved LPS-induced intestinal barrier permeability reduction and transepithelial resistance. Furthermore, HK-LP 6105, 6115, and 6235 inhibited the pro-inflammatory factors (TNF-α, IL-1ß, IL-6) and increased the expression of the anti-inflammatory factors (IL-4, IL-10, and TGF-ß). HK-LP 6105, 6115, and 6235 ameliorated the inflammatory response. It inhibited the nuclear factor kappa B (NF-κB) signaling pathway-mediated myosin light chain (MLC)/MLC kinase signaling pathway by downregulating the Toll-like receptor 4 (TLR4)/NF-κB pathway. Thus, the results suggest that HK-LP 6150, 6115, and 6235 may improve intestinal health by regulating inflammation and TJ proteins. Postbiotics produced by these strains exhibit anti-inflammatory properties that can protect the intestinal barrier.
Assuntos
Lipopolissacarídeos , NF-kappa B , Humanos , NF-kappa B/metabolismo , Células CACO-2 , Lipopolissacarídeos/farmacologia , Cadeias Leves de Miosina , Lacticaseibacillus , Mucosa Intestinal/metabolismo , Temperatura Alta , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Fosforilação , Anti-Inflamatórios/farmacologia , Junções Íntimas/metabolismoRESUMO
Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 µg/kg diet.
Assuntos
Carpas , Poluentes Químicos da Água , Animais , NF-kappa B/metabolismo , Suplementos Nutricionais/análise , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Aflatoxina B1/toxicidade , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Carpas/metabolismo , Brânquias/metabolismo , Imunidade Inata , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Poluentes Químicos da Água/toxicidade , Transdução de Sinais , Dieta/veterinária , Antioxidantes/metabolismo , Glutationa , Ração Animal/análiseRESUMO
Mechanical properties of healthy and Dupuytren fibroblasts were investigated by atomic force microscopy (AFM). In addition to standard force curves, rheological properties were assessed using an oscillatory testing methodology, in which the frequency was swept from 1 Hz to 1 kHz, and data were analyzed using the structural damping model. Dupuytren fibroblasts showed larger apparent Young's modulus values than healthy ones, which is in agreement with previous results. Moreover, cell mechanics were compared before and after ML-7 treatment, which is a myosin light chain kinase inhibitor (MLCK) that reduces myosin activity and hence cell contraction. We employed two different concentrations of ML-7 inhibitor and could observe distinct cell reactions. At 1 µM, healthy and scar fibroblasts did not show measurable changes in stiffness, but Dupuytren fibroblasts displayed a softening and recovery after some time. When increasing ML-7 concentration (3 µM), the majority of cells reacted, Dupuytren fibroblasts were the most susceptible, not being able to recover from the drug and dying. These results suggested that ML-7 is a potent inhibitor for MLCK and that myosin II is essential for cytoskeleton stabilization and cell survival.
Assuntos
Citoesqueleto , Contratura de Dupuytren , Fibroblastos , Microscopia de Força Atômica , Contração Muscular , Cadeias Leves de Miosina , Humanos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Contratura de Dupuytren/tratamento farmacológico , Contratura de Dupuytren/metabolismo , Contratura de Dupuytren/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fenômenos Mecânicos , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Quinase de Cadeia Leve de Miosina/uso terapêutico , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologiaRESUMO
Advanced glycation end products (AGEs) disturb endothelial barrier function and contribute to age-related diseases. As microRNAs (miRNAs) are potential therapeutic agents, targeting AGEs-associated signaling using miRNAs in endothelial cells may be an effective intervention strategy for age-related vascular disorders. This study investigated the effects of AGEs on the endothelial cell senescence and barrier function in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with AGEs and transfected with miRNA-1-3p mimics to induce overexpression of miR-1-3p. Senescence-associated ß-galactosidase (SA-ß-Gal) staining and senescence-related proteins P53, P21, and P16 were detected to evaluate the endothelial cell senescence. The expression levels of myosin light chain kinase (MLCK) signaling and transendothelial electric resistance (TEER) were used to indicate endothelial barrier function. AGEs significantly increased SA-ß-gal staining-positive cells accompanied by the upregulation of P53, P21, and P16 expression. AGEs also damaged endothelial barrier function by decreasing TEER and increasing zonula occludens protein 1, p-MLC/MLC, and MLCK. miRNA-1-3p was significantly reduced in HUVECs treated with AGEs. miR-1-3p overexpression decreased MLCK signal and improved AGEs-induced endothelial barrier function impairment. Meanwhile, miR-1-3p overexpression ameliorated oxidative stress and endothelial cell senescence induced by AGEs. AGEs induced endothelial cell senescence and endothelial barrier dysfunction by regulating miR-1-3p/MLCK signaling pathway.
Assuntos
MicroRNAs , Quinase de Cadeia Leve de Miosina , Humanos , Senescência Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , MicroRNAs/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Produtos Finais de Glicação Avançada/metabolismoRESUMO
RATIONALE: Intestinal permeability plays an important role in gut-brain axis communication. Recent studies indicate that intestinal permeability increases in neonate pups during maternal separation (MS). OBJECTIVES: The present study aims to determine whether pharmacological inhibition of myosin light chain kinase (MLCK), which regulates tight junction contraction and controls intestinal permeability, in stressed neonates, protects against the long-term effects of MS. METHODS: Male Wistar rats were exposed to MS (3 h per day from post-natal day (PND)2 to PND14) or left undisturbed and received daily intraperitoneal injection of a MLCK inhibitor (ML-7, 5 mg/kg) or vehicle during the same period. At adulthood, emotional behaviors, corticosterone response to stress, and gut microbiota composition were analyzed. RESULTS: ML-7 restored gut barrier function in MS rats specifically during the neonatal period. Remarkably, ML-7 prevented MS-induced sexual reward-seeking impairment and reversed the alteration of corticosterone response to stress at adulthood. The effects of ML-7 were accompanied by the normalization of the abundance of members of Lachnospiraceae, Clostridiales, Desulfovibrio, Bacteroidales, Enterorhabdus, and Bifidobacterium in the feces of MS rats at adulthood. CONCLUSIONS: Altogether, our work suggests that improvement of intestinal barrier defects during development may alleviate some of the long-term effects of early-life stress and provides new insight on brain-gut axis communication in a context of stress.
Assuntos
Azepinas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Privação Materna , Naftalenos/farmacologia , Estresse Psicológico/metabolismo , Animais , Animais Recém-Nascidos , Azepinas/uso terapêutico , Corticosterona/metabolismo , Relação Dose-Resposta a Droga , Feminino , Microbioma Gastrointestinal/fisiologia , Masculino , Quinase de Cadeia Leve de Miosina/farmacologia , Quinase de Cadeia Leve de Miosina/uso terapêutico , Naftalenos/uso terapêutico , Gravidez , Ratos , Ratos Wistar , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/psicologia , Fatores de TempoRESUMO
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/farmacologiaRESUMO
Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca(2+) sensitivity of force (pCa(50)), PKA treatment has been shown to decrease pCa(50), presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca(2+)-independent force and maximum Ca(2+)-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I(1,1)/I(1,0)) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d(1,0)) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I(1,1)/I(1,0) and, as hypothesized, treatment with MLCK also increased I(1,1)/I(1,0), which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by 2 nm (3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca(2+) sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.
Assuntos
Proteínas de Transporte/fisiologia , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/fisiologia , Função Ventricular Direita/fisiologia , Animais , Cálcio/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Animais , Contração Miocárdica/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Função Ventricular Direita/efeitos dos fármacosRESUMO
As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK((1-320))-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC(20) and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Domínio Catalítico , Galinhas , Proteínas Quinases Associadas com Morte Celular , Ativação Enzimática , Íleo/enzimologia , Técnicas In Vitro , Microcistinas/farmacologia , Proteínas Musculares/metabolismo , Músculo Liso/efeitos dos fármacos , Mutação , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 1/metabolismo , Ratos , Especificidade por SubstratoRESUMO
A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73+/-9), the ratio was approximately 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at approximately 1 nM free [Ca(2+)]. There were two MLCK pools that bound unphosphorylated SMM with K(d) approximately 10 and 0.2 microM and phosphorylated SMM with K(d) approximately 20 and 0.2 microM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca(2+) and phosphorylation of SMM occurred at a pCa(50) of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.
Assuntos
Calmodulina/metabolismo , Complexos Multiproteicos/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas de Músculo Liso/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Galinhas , Ensaio de Imunoadsorção Enzimática , Humanos , Cinética , Magnésio/farmacologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de ProteínaRESUMO
Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional 45Ca2+ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP3-induced Ca2+ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3-induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.
Assuntos
Canais de Cálcio/metabolismo , Calmodulina/metabolismo , Calmodulina/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Sítios de Ligação , Células COS , Cálcio/análise , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Pulmão/citologia , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/farmacologia , Oócitos/efeitos dos fármacos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Ouriços-do-Mar/citologia , Spodoptera/citologia , Spodoptera/genéticaRESUMO
It is generally recognized that ventricular myosin regulatory light chains (RLC) are approximately 40% phosphorylated under basal conditions, and there is little change in RLC phosphorylation with agonist stimulation of myocardium or altered stimulation frequency. To establish the functional consequences of basal RLC phosphorylation in the heart, we measured mechanical properties of rat skinned trabeculae in which approximately 7% or approximately 58% of total RLC was phosphorylated. The protocol for achieving approximately 7% phosphorylation of RLC involved isolating trabeculae in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC from its baseline level. Subsequent phosphorylation to approximately 58% of total was achieved by incubating BDM-treated trabeculae in solution containing smooth muscle myosin light chain kinase, calmodulin, and Ca2+ (i.e., MLCK treatment). After MLCK treatment, Ca2+ sensitivity of force increased by 0.06 pCa units and maximum force increased by 5%. The rate constant of force development (ktr) increased as a function of Ca2+ concentration in the range between pCa 5.8 and pCa 4.5. When expressed versus pCa, the activation dependence of ktr appeared to be unaffected by MLCK treatment; however, when activation was expressed in terms of isometric force-generating capability (as a fraction of maximum), MLCK treatment slowed ktr at submaximal activations. These results suggest that basal phosphorylation of RLC plays a role in setting the kinetics of force development and Ca2+ sensitivity of force in cardiac muscle. Our results also argue that changes in RLC phosphorylation in the range examined here influence actin-myosin interaction kinetics differently in heart muscle than was previously reported for skeletal muscle.
Assuntos
Cálcio/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Feminino , Técnicas In Vitro , Cinética , Quinase de Cadeia Leve de Miosina/farmacologia , Fosforilação , Ratos , Ratos WistarRESUMO
Telokin, a 17 kDa smooth muscle specific protein, consists of the C-terminal domain of MLCK, is phosphorylated by PKA and PKG at Ser13 in vivo (Wu et al. (1998) J Biol Chem 273: 11362-11369; Walker et al. (2001) J. Biol Chem 276: 24519-24524) and is proposed to induce Ca2+-desensitization through activation of myosin phosphatase (Wu et al. (1998) J. Biol Chem 273: 11362-11369). Telokin is reported to be highly expressed in phasic with only trace amounts in tonic smooth muscle. In alpha-toxin permeabilized femoral artery, 5 microM 8-Br-cGMP induced a two-fold increase in telokin phosphorylation and a maximal 30% relaxation of Ca2+-activated force compared to a 90% relaxation in phasic ileum muscle consistent with the relative amounts of telokin expressed in ileum, 27+/-4.6 microM SEM compared to 6+/-1.7 microM SEM, in femoral artery. Recombinant Wt telokin and the phospho-telokin mutant, S13D relaxed telokin-depleted femoral artery, by 38+/-8% SEM and 60+/-20% SEM, respectively. 8-Br-cGMP increased the rate and decreased the amplitude of force development initiated by photolysis of caged ATP in alpha-toxin permeabilized ileum and femoral artery smooth muscle, consistent with a cGMP-induced increase in phosphatase activity. Similarly, in telokin depleted ileum, recombinant S13D mutant telokin significantly increased the rate (0.08+/-0.01 s-1 vs. 014+/-0.02 s-1) and decreased force amplitude. In conclusion, our data support a role for telokin in cyclic nucleotide-induced relaxation of not only phasic, but also tonic smooth muscle and that this relaxation is mediated by activation of myosin phosphatase activity leading to a decrease in myosin light chain phosphorylation.
Assuntos
Cálcio/fisiologia , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Peptídeos/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Ativação Enzimática , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/enzimologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Quinase de Cadeia Leve de Miosina/biossíntese , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/farmacologia , Fragmentos de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosforilação , Coelhos , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
Combretastatin A-4-phosphate (CA-4-P) is a tubulin-binding compound currently in clinical trial as a tumor vascular-targeting agent. In endothelial cells, CA-4-P is known to cause microtubule depolymerization, but little is known about its subsequent effects on cell morphology and function. Here, we demonstrate that within minutes of endothelial cell exposure to CA-4-P, myosin light chain (MLC) was phosphorylated, leading to actinomyosin contractility, assembly of actin stress fibers, and formation of focal adhesions. These cytoskeletal alterations appeared to be a consequence of Rho activation, as they were abolished by either the Rho inhibitor C3 exoenzyme or Rho-kinase inhibitor Y-27632. In response to CA-4-P, some cells rapidly assumed a blebbing morphology in which F-actin accumulated around surface blebs, stress fibers misassembled into a spherical network surrounding the cytoplasm, and focal adhesions appeared malformed. Blebbing was associated with decreased cell viability and could be inhibited by Rho/Rho-kinase inhibitors or by blocking the CA-4-P-mediated activation of stress-activated protein kinase-2/p38. The extracellular-regulated kinases 1 and 2 (ERK-1/2) were shown to protect against blebbing since blebbing was attenuated on ERK-1/2 stimulation and was up-regulated by specific inhibition of ERK-1/2 activation. The use of MLC kinase (MLCK) and myosin adenosine triphosphatase inhibitors led us to propose a role for MLCK and myosin activity independent of MLC phosphorylation in regulating the blebbing process. CA-4-P-mediated contractility and blebbing were associated with a Rho-dependent increase in monolayer permeability to dextrans, suggesting that such functional changes may be important in the rapid response of the tumor endothelium to CA-4-P in vivo.
Assuntos
Actinas/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Citoesqueleto/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/ultraestrutura , Estilbenos/farmacologia , Actinas/metabolismo , Proteínas de Fase Aguda/efeitos dos fármacos , Proteínas de Fase Aguda/farmacologia , Proteínas de Fase Aguda/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Endotélio Vascular/citologia , Adesões Focais/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/farmacologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Quinase de Cadeia Leve de Miosina/farmacologia , Quinase de Cadeia Leve de Miosina/fisiologia , Miosinas/farmacologia , Miosinas/fisiologia , Necrose , Fibras de Estresse/efeitos dos fármacosRESUMO
The postsynaptic density (PSD) at excitatory dendritic synapses comprises a protein complex of glutamate receptors, scaffolding elements, and signaling enzymes. For example, NMDA receptors (NMDARs) are linked to several proteins in the PSD, such as PSD-95, and are also tethered via binding proteins such as alpha-actinin directly to filamentous actin of the cytoskeleton. Depolymerization of the cytoskeleton modulates the activity of NMDARs, and, in turn, strong activation of NMDARs can trigger depolymerization of actin. Myosin, the motor protein of muscular contraction and nonmuscle motility, is also associated with NMDARs and the PSD. We show here that constitutively active myosin light chain kinase (MLCK) enhances NMDAR-mediated whole-cell and synaptic currents in acutely isolated CA1 pyramidal and cultured hippocampal neurons, whereas inhibitors of MLCK depress these currents. This MLCK-dependent regulation was observed in cell-attached patches but was lost after excision to inside-out patches. Furthermore, the enhancement induced by constitutively active MLCK and the depression of MLCK inhibitors were eliminated after depolymerization of the cytoskeleton. NMDARs and MLCK did not colocalize in clusters on the dendrites of cultured hippocampal neurons, further indicating that the effects of MLCK are mediated indirectly via actomyosin. Our results suggest that MLCK enhances actomyosin contractility to either increase the membrane tension on NMDARs or to alter physical relationships between the actin cytoskeleton and the linker proteins of NMDARs.
Assuntos
Actinas/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Carbocianinas , Separação Celular , Células Cultivadas , Dendritos/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes , Hipocampo , Camundongos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/farmacologia , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
To understand how the plasma membrane Ca(2+) pump (PMCA) behaves under changing Ca(2+) concentrations, it is necessary to obtain information about the Ca(2+) dependence of the rate constants for calmodulin activation (k(act)) and for inactivation by calmodulin removal (k(inact)). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k(act) increased with Ca(2+) along a sigmoidal curve. At all Ca(2+) concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca(2+) concentration and also tested these simulations experimentally; PMCA was activated by 500 nm Ca(2+) and then exposed to 50 nm Ca(2+) for 10 to 150 s, and then Ca(2+) was increased again to 500 nm. During the second exposure to 500 nm Ca(2+), the activity reached steady state faster than during the first exposure at 500 nm Ca(2+). This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nm Ca(2+). The peptide inhibited the activity of PMCA2b when the exposure to 50 nm Ca(2+) was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca(2+). The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca(2+) more quickly in subsequent spikes after an initial activating spike.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/enzimologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Transporte de Cátions , Humanos , Cinética , Modelos Teóricos , Quinase de Cadeia Leve de Miosina/farmacologia , Fragmentos de Peptídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática , Isoformas de Proteínas , Proteínas Recombinantes/metabolismoRESUMO
In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.
Assuntos
Junção Esofagogástrica/fisiologia , Esôfago/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Quinase de Cadeia Leve de Miosina/farmacologia , Proteína Quinase C/fisiologia , Acetilcolina/farmacologia , Animais , Proteínas de Ligação ao Cálcio/farmacologia , Calmodulina/fisiologia , Proteínas de Ligação a Calmodulina/farmacologia , Gatos , Células Cultivadas , Feminino , Masculino , Proteínas dos Microfilamentos , Músculo Liso/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Transdução de Sinais , CalponinasRESUMO
Although the signaling pathways leading to hydrogen peroxide (H2O2)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2 transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H2O2 induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H2O2-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton.
Assuntos
Citoesqueleto/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Actinas/metabolismo , Actinas/fisiologia , Animais , Bovinos , Linhagem Celular , Citoesqueleto/enzimologia , Citoesqueleto/fisiologia , Endotélio Vascular/enzimologia , Quinase de Cadeia Leve de Miosina/farmacologia , Miosinas/metabolismo , Miosinas/fisiologia , Proteína Quinase C/antagonistas & inibidores , Artéria PulmonarRESUMO
Actions of myosin light chain kinase inhibitors were tested on delayed rectifier potassium current (IK) in dissociated bullfrog sympathetic neurons. A microbial product, wortmannin (10 microM, extracellularly) and a synthetic peptide, SM-1 (20 microM, intracellularly) caused approximately 35 mV hyperpolarizing shift of the inactivation curve. Substitution of ATP (1.15 mM) in the pipette solution with 5'-adenylylimidodiphosphate mimicked the actions of wortmannin and SM-1. Results suggest that phosphorylation of myosin may modulate kinetics for the inactivation of IK.
Assuntos
Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Neurônios/metabolismo , Canais de Potássio/metabolismo , Sistema Nervoso Simpático/metabolismo , Adenilil Imidodifosfato/farmacologia , Androstadienos/farmacologia , Animais , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Gânglios Simpáticos/citologia , Gânglios Simpáticos/efeitos dos fármacos , Técnicas In Vitro , Quinase de Cadeia Leve de Miosina/farmacologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Canais de Potássio/efeitos dos fármacos , Rana catesbeiana , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/efeitos dos fármacos , WortmaninaRESUMO
Myosin light chain kinase (MLCK) is present in muscle cells including those of smooth muscle as an actin-binding protein. By avoiding complication introduced as a result of kinase activity of MLCK, we have demonstrated regulatory role of MLCK through its actin-binding activity [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. To analyze such a regulatory role of MLCK, we compared the effects of MLCK on the velocity of the movement of actin filaments on a surface coated with smooth muscle myosin with those of another actin-binding proteins in smooth muscle, namely, caldesmon (CaD) and calponin (CaP). Both CaD and CaP stimulated movement when their concentrations were low, but they inhibited movement as their concentrations were increased. Calmodulin (CaM) in the presence of Ca2+ (Ca-CaM) antagonized the inhibition but hardly affected the stimulation. The effect of MLCK, by contrast, was simply inhibitory when Ca-CaM was not present. No stimulation was observed until Ca-CaM was added. The inhibitory ability of these actin-binding proteins increased in the following order: CaD < CaP < MLCK. The effect of MLCK and CaD on movement was further examined on surfaces coated with skeletal muscle myosin. The basic effect was similar to that observed with smooth muscle myosin. However, 10-fold greater concentrations of MLCK and CaD were required for a comparable effect. Such an increase in the required concentration was also observed when the velocity of movement was increased by elevation of the temperature during the assay with smooth muscle myosin. Thus, it is the velocity of movement itself that determines the required concentrations of MLCK and CaD.