共查询到20条相似文献,搜索用时 31 毫秒
1.
Zhou YW Oak SA Senogles SE Jarrett HW 《American journal of physiology. Cell physiology》2005,288(2):C377-C388
-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gs and G occurs and active Gs, measured as GTP-35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gs is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-S-bound) Gs, and the Ca2+ channel is inhibited by laminin-1. The laminin 1-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain 相似文献
2.
Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2 总被引:3,自引:0,他引:3
We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC20) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P1 and S1P2 receptors. S1P activated Gq, G13, and all Gi isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or Gq antibody, PLC-1 or PLC-3 antibody, and by expression of Gq or Gi minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G13 or Gq minigene and abolished by expression of both. S1P stimulated Ca2+ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC50 1 nM). Initial contraction and MLC20 phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Gq or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC20 phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P2 and S1P1 involving concurrent activation of PLC-1 and PLC-3 via Gq and Gi, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca2+ release and MLCK-mediated MLC20 phosphorylation, and 2) sustained contraction exclusively mediated by S1P2 involving activation of RhoA via Gq and G13, resulting in Rho kinase- and PKC-dependent MLC20 phosphorylation. muscle contraction; signal transduction 相似文献
3.
Sade H Muraki K Ohya S Hatano N Imaizumi Y 《American journal of physiology. Cell physiology》2006,290(1):C77-C86
We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the -subunit of the BK channel (BK-), both - and 1-subunits (BK-1), or both - and 4-subunits (BK-4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-1 within a similar concentration range. Because methAEA could potentiate BK-, BK-1, and BK-4 with similar efficacy, the -subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK- and HEK-BK-1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed. anandamide; channel opener 相似文献
4.
CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake 总被引:1,自引:0,他引:1
Codina Juan; Li Jian; DuBose Thomas D. Jr. 《American journal of physiology. Cell physiology》2005,288(6):C1279
The carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK2, we selected 84 amino acids in the CT of the -subunit of mouse colonic H+-K+-ATPase (CT-HK2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK2 with CD63, recombinant CT-HK2 and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK2 protein (but not CT-HK1) coprecipitated with CD63, confirming stable assembly of HK2 with CD63. In HEK-293 transfected with HK2 plus 1-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK2/NK1 and 86Rb+ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK2-NK1 complex in the cell membrane. protein assembly; cell surface localization 相似文献
5.
Feng S Lu X Reséndiz JC Kroll MH 《American journal of physiology. Cell physiology》2006,291(6):C1346-C1354
Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its -subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin IIb3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on IIb3, we examined IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain, and Syk coimmunoprecipitate with IIb3 in resting platelets and that 120 dyn/cm2 shear stress leads to their disassociation from IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the 3 tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to IIb3. Syk's disassociation from 3 is inhibited when VWF binding to either GpIb-IX-V or IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to IIb3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing IIb3 with 3 truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates IIb3 function and suggest that shear-induced IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the 3-tail. platelets; mechanoreceptor; integrin; shear stress; signal transduction 相似文献
6.
Mechanical loading and injury induce human myotubes to release neutrophil chemoattractants 总被引:1,自引:0,他引:1
Tsivitse SK Mylona E Peterson JM Gunning WT Pizza FX 《American journal of physiology. Cell physiology》2005,288(3):C721-C729
The purpose of this study was to 1) test the hypothesis that skeletal muscle cells (myotubes) after mechanical loading and/or injury are a source of soluble factors that promote neutrophil chemotaxis and superoxide anion (O2·) production and 2) determine whether mechanical loading and/or injury causes myotubes to release cytokines that are known to influence neutrophil responses [tumor necrosis factor- (TNF-), IL-8, and transforming growth factor-1 (TGF-1)]. Human myotubes were grown in culture and exposed to either a cyclic strain (0, 5, 10, 20, or 30% strain) or a scrape injury protocol. Protocols of 5, 10, and 20% strain did not cause injury, whereas 30% strain and scrape injury caused a modest and a high degree of injury, respectively. Conditioned media from strained myotubes promoted chemotaxis of human blood neutrophils and primed them for O2· production in a manner that was dependent on a threshold of strain and independent from injury. Neutrophil chemotaxis, but not priming, progressively increased with higher magnitudes of strain. Conditioned media only from scrape-injured myotubes increased O2· production from neutrophils. Concentrations of IL-8 and total TGF-1 in conditioned media were reduced by mechanical loading, whereas TNF- and active TGF-1 concentrations were unaffected. In conclusion, skeletal muscle cells after mechanical loading and injury are an important source of soluble factors that differentially influence neutrophil chemotaxis and the stages of neutrophil-derived reactive oxygen species production. Neutrophil responses elicited by mechanical loading, however, did not parallel changes in the release of IL-8, TGF-1, or TNF- from skeletal muscle cells. inflammation; cytokines; exercise; free radicals 相似文献
7.
Xiao Q Zeng L Zhang Z Hu Y Xu Q 《American journal of physiology. Cell physiology》2007,292(1):C342-C352
Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1+) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1+ progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 690 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1+ progenitors with antibodies against integrin 1, v, and 1, but not 3, inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor- significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (1, 1, and v)-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor- signaling pathways are involved in SMC differentiation. progenitor cells; extracellular matrix; growth factor receptors; platelet-derived growth factor 相似文献
8.
Smallwood ND Hausman BS Wang X Liedtke CM 《American journal of physiology. Cell physiology》2005,288(4):C906-C912
Direct binding of nonmuscle F-actin and the C2-like domain of PKC- (C2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC- with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three NH2-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging from 1.2 to 2.2 nmol (611 µM). NH2-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the C2-like domain encodes a 1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the 1-sheet region of the C2-like domain in airway epithelial cells. truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter 相似文献
9.
Matrix metalloproteinases mediate beta-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes 总被引:1,自引:0,他引:1
Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in -adrenergic receptor (-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). -AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed -AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited -AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with 1-integrins after -AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of 1-integrin signaling using laminin inhibited the increased association of MMP-2 with 1-integrins. -AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) -AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits -AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with 1 integrins and poly-ADP-ribose-polymerase cleavage. integrins; poly-ADP-ribose-polymerase 相似文献
10.
Laminin-10 and Lutheran blood group glycoproteins in adhesion of human endothelial cells 总被引:3,自引:0,他引:3
Vainionpää N Kikkawa Y Lounatmaa K Miner JH Rousselle P Virtanen I 《American journal of physiology. Cell physiology》2006,290(3):C764-C775
Laminin 5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of 5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin 5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed Mr 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins 1 and v3, whereas in the adhesion to laminin-10/11, Lu and integrin 1 were involved. In the cells adhering to the 5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that 5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to 5 laminins in collaboration with integrins 1 and v3. integrin; cycloheximide 相似文献
11.
Uezono Y Kaibara M Murasaki O Taniyama K 《American journal of physiology. Cell physiology》2004,287(4):C885-C894
We studied the functions of -subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl currents in oocytes expressing 2-adrenoceptor and the protein kinase A-dependent Cl channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala2, D-Leu5]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing 2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), -opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (Gt). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or -opioid receptor alone. They elicited Ca2+-activated Cl currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of Gt, suggesting that -subunits of Gi/o protein activate PLC-2 and then cause intracellular Ca2+ mobilization. Our results indicate that -subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology 相似文献
12.
Oshikawa J Toya Y Fujita T Egawa M Kawabe J Umemura S Ishikawa Y 《American journal of physiology. Cell physiology》2003,285(3):C567-C574
Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR 7-subunit has a property of high Ca2+ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR7 existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR 5- and 2-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR7. Cholesterol depletion from plasma membranes with methyl--cyclodextrin redistributed nAChR7 and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with -bungarotoxin, a specific antagonist of nAChR7, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR7 has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR7 with AC within plasma membranes. In addition, nAChR7 may regulate the AC activity via Ca2+ within lipid rafts. cholesterol; PC-12 cells 相似文献
13.
Berfield AK Hansen KM Abrass CK 《American journal of physiology. Cell physiology》2006,291(4):C589-C599
Temporal and spatial differences in extracellular matrix play critical roles in cell proliferation, differentiation and migration. Different migratory stimuli use different substrates and receptors to achieve cell migration. To understand the mechanism of insulin-like growth factor binding protein-5 (IGFBP-5)-induced migration in mesangial cells, the roles of integrins and substrates were examined. IGFBP-5 induced an increase in mRNA expression for laminin (LN) chains lama4, lamb2, and lamc1, suggesting that LN-9 might be required for migration. Antibodies to the LN4 and LN2 chains, but not LN1, blocked IGFBP-5-induced migration. Anti-sense morpholino oligonucleotide inhibition of expression of LN4 substantially reduced expression of LN-8/9 (411/421, 411/421) and prevented IGFBP-5-induced migration. Anti-sense inhibition of lamb2 reduced expression of LN-9. Absence of LN-9 prevented IGFBP-5-induced migration, which was not preserved by continued expression of LN-8. The requirement for LN-9 was further supported by studies of T98G cells, which express predominantly LN-8. IGFBP-5 had little effect on migration in these cells, but increased migration when T98G cells were plated on LN-8/9. IGFBP-5-mediated mesangial cell migration was inhibited by antibodies that block attachment to 61-integrins but was unaffected by antibodies and disintegrins that block binding to other integrins. Furthermore, in cells with anti-sense inhibited expression of LN-9, integrin 61 was no longer detected on the cell surface. These studies suggest the specificity of mechanisms of migration induced by specific stimuli and for the first time demonstrate a unique function for LN-9 in mediating IGFBP-5-induced migration. migration; integrins; extracellular matrix 相似文献
14.
Lopez MA Mayer U Hwang W Taylor T Hashmi MA Jannapureddy SR Boriek AM 《American journal of physiology. Cell physiology》2005,288(2):C282-C289
71 integrin is a transmembrane structural and receptor protein of skeletal muscles, and the absence of 7-integrin causes muscular dystrophy. We hypothesized that the absence of 7-integrin alters compliance and viscoelasticity and disrupts the mechanical coupling between passive transverse and axial contractile elements in the diaphragm. In vivo the diaphragm is loaded with pressure, and therefore axial and transverse length-tension relationships are important in assessing its function. We determined diaphragm passive length-tension relationships and the viscoelastic properties of its muscle in 1-month-old 7-integrin-null mice and age-matched controls. Furthermore, we measured the isometric contractile properties of the diaphragm from mutant and normal mice in the absence and presence of passive force applied in the transverse direction to fibers in 1-month-old and 5-month-old mutant mice. We found that compared with controls, the diaphragm direction of 7-integrin-null mutants showed 1) a significant decrease in muscle extensibility in 1-year-old mice, whereas muscle extensibility increased in the 1-month-old mice; 2) altered muscle viscoelasticity in the transverse direction of the muscle fibers of 1-month-old mice; 3) a significant increase in force-generating capacity in the diaphragms of 1-month-old mice, whereas in 5-month-old mice muscle contractility was depressed; and 4) significant reductions in mechanical coupling between longitudinal and transverse properties of the muscle fibers of 1-month-old mice. These findings suggest that 7-integrin serves an important mechanical function in the diaphragm by contributing to passive compliance, viscoelasticity, and modulation of its muscle contractile properties. muscular dystrophy; respiratory muscles; transmembrane proteins 相似文献
15.
Activation of PLC-delta1 by Gi/o-coupled receptor agonists 总被引:1,自引:0,他引:1
Murthy KS Zhou H Huang J Pentyala SN 《American journal of physiology. Cell physiology》2004,287(6):C1679-C1687
The mechanism of phospholipase (PLC)- activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca2+ stimulated an eightfold increase in PLC-1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three Gi/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen2,D-Pen5]enkephalin, and A1 agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-1(E341R/D343R; 6576%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca2+ influx and was not observed in the absence of extracellular Ca2+, but was partly inhibited by nifedipine (1630%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca2+ channels. Treatment of the cells with a Gq/13-coupled receptor agonist, CCK-8, caused only transient, PLC-1-mediated PI hydrolysis. Unlike Gi/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G13 minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca2+ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (25%) and strongly inhibited by SKF-96365 (75%) and in cells expressing PLC-1(E341R/D343R). Agonist-independent Ca2+ release or Ca2+ influx via voltage-gated Ca2+ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-1 antibody or nifedipine. We conclude that PLC-1 is activated by Gi/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca2+ influx via store-operated Ca2+ channels. phospholipase C; G protein 相似文献
16.
Wilson L Yang Q Szustakowski JD Gullicksen PS Halse R 《American journal of physiology. Cell physiology》2007,292(5):C1599-C1605
Oxidative cells increase mitochondrial mass in response to stimuli such as changes in energy demand or cellular differentiation. This plasticity enables the cell to adapt dynamically to achieve the necessary oxidative capacity. However, the pathways involved in triggering mitochondrial biogenesis are poorly defined. The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells. C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 h. Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate, and maximal oxidative capacity. The increase in mitochondrial biogenesis was independent of increases in peroxisomal proliferator activator receptor- coactivator-1 (PGC-1) and PGC-1 mRNA expression. To further assess whether PGC-1 expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1 before treatment with pyruvate. Despite a 70% reduction in PGC-1 mRNA, the effect of pyruvate was preserved. Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1 null mice. These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity. oxidative metabolism; peroxisomal proliferator activator receptor- coactivator-1, mitochondria; muscle 相似文献
17.
Ju JS Gitcho MA Casmaer CA Patil PB Han DG Spencer SA Fisher JS 《American journal of physiology. Cell physiology》2007,292(1):C564-C572
Data from the use of activators and inhibitors of the AMP-activated protein kinase (AMPK) suggest that AMPK increases sensitivity of glucose transport to stimulation by insulin in muscle cells. We assayed insulin action after adenoviral (Ad) transduction of constitutively active (CA; a truncated form of AMPK1) and dominant-negative (DN; which depletes endogenous AMPK) forms of AMPK (Ad-AMPK-CA and Ad-AMPK-DN, respectively) into C2C12 myotubes. Compared with control (Ad-green fluorescent protein), Ad-AMPK-CA increased the ability of insulin to stimulate glucose transport. The increased insulin action in cells expressing AMPK-CA was suppressed by compound C (an AMPK inhibitor). Exposure of cells to 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (an AMPK activator) increased insulin action in uninfected myotubes and myotubes transduced with green fluorescent protein but not in Ad-AMPK-DN-infected myotubes. In Ad-AMPK-CA-transduced cells, serine phosphorylation of insulin receptor substrate 1 was decreased at a mammalian target of rapamycin (or p70 S6 kinase) target site that has been reported to be associated with insulin resistance. These data suggest that, in myotubes, activated AMPK1 is sufficient to increase insulin action and that the presence of functional AMPK is required for 5-aminoimidazole-4-carboxamide-1,D-ribofuranoside-related increases in insulin action. compound C; AMPK; insulin sensitivity; Akt; mTOR 相似文献
18.
Menon B Singh M Ross RS Johnson JN Singh K 《American journal of physiology. Cell physiology》2006,290(1):C254-C261
Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with 1 integrins. Herein we tested the hypothesis that MMP-2 impairs 1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of 1 integrins using adenoviruses expressing the human 1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of 1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the 1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH2-terminal kinase; cytochrome c 相似文献
19.
van der Velden JL Langen RC Kelders MC Wouters EF Janssen-Heininger YM Schols AM 《American journal of physiology. Cell physiology》2006,290(2):C453-C462
Skeletal muscle atrophy is a prominent and disabling feature of chronic wasting diseases. Prevention or reversal of muscle atrophy by administration of skeletal muscle growth (hypertrophy)-stimulating agents such as insulin-like growth factor I (IGF-I) could be an important therapeutic strategy in these diseases. To elucidate the IGF-I signal transduction responsible for muscle formation (myogenesis) during muscle growth and regeneration, we applied IGF-I to differentiating C2C12 myoblasts and evaluated the effects on phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3 (GSK-3) signaling and myogenesis. IGF-I caused phosphorylation and inactivation of GSK-3 activity via signaling through the PI3K/Akt pathway. We assessed whether pharmacological inhibition of GSK-3 with lithium chloride (LiCl) was sufficient to stimulate myogenesis. Addition of IGF-I or LiCl stimulated myogenesis, evidenced by increased myotube formation, muscle creatine kinase (MCK) activity, and troponin I (TnI) promoter transactivation during differentiation. Moreover, mRNAs encoding MyoD, Myf-5, myogenin, TnI-slow, TnI-fast, MCK, and myoglobin were upregulated in myoblasts differentiated in the presence of IGF-I or LiCl. Importantly, blockade of GSK-3 inhibition abrogated IGF-I- but not LiCl-dependent stimulation of myogenic mRNA accumulation, suggesting that the promyogenic effects of IGF-I require GSK-3 inactivation and revealing an important negative regulatory role for GSK-3 in myogenesis. Therefore, this study identifies GSK-3 as a potential target for pharmacological stimulation of muscle growth. insulin-like growth factor I; muscle hypertrophy 相似文献
20.
Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell 相似文献