scholarly journals Effect of Elevated Intracellular cAMP Levels on Actomyosin Contraction in Bovine Trabecular Meshwork Cells

2011 ◽  
Vol 52 (3) ◽  
pp. 1474 ◽  
Author(s):  
Charanya Ramachandran ◽  
Rajkumar V. Patil ◽  
Najam A. Sharif ◽  
Sangly P. Srinivas
Keyword(s):  
Trabecular Meshwork ◽  
Intracellular Camp ◽  
Trabecular Meshwork Cells ◽  
Actomyosin Contraction ◽  
Camp Levels

scholarly journals MRP4-Mediated Regulation of Intracellular cAMP and cGMP Levels in Trabecular Meshwork Cells and Homeostasis of Intraocular Pressure

2013 ◽  
Vol 54 (3) ◽  
pp. 1636 ◽  
Author(s):  
Padmanabhan P. Pattabiraman ◽  
Paula E. Pecen ◽  
Ponugoti Vasantha Rao
Keyword(s):  
Intraocular Pressure ◽  
Trabecular Meshwork ◽  
Intracellular Camp ◽  
Trabecular Meshwork Cells ◽  
Camp And Cgmp

scholarly journals Down-regulation of 14-3-3 Zeta Inhibits TGF-β1–Induced Actomyosin Contraction in Human Trabecular Meshwork Cells Through RhoA Signaling Pathway

2016 ◽  
Vol 57 (2) ◽  
pp. 719 ◽  
Author(s):  
Yiming Ye ◽  
Yangfan Yang ◽  
Xiaoxiao Cai ◽  
Liling Liu ◽  
Kaili Wu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Trabecular Meshwork ◽  
Down Regulation ◽  
Trabecular Meshwork Cells ◽  
Rhoa Signaling ◽  
Actomyosin Contraction ◽  
Tgf Β1

Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly

2004 ◽  
Vol 287 (1) ◽  
pp. L86-L93 ◽  
Author(s):  
Anna A. Birukova ◽  
Feng Liu ◽  
Joe G. N. Garcia ◽  
Alexander D. Verin
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Fiber Formation ◽  
Regulatory Subunit ◽  
Intracellular Camp ◽  
Protective Effects ◽  
Barrier Dysfunction ◽  
Mlc Phosphorylation ◽  
Actomyosin Contraction ◽  
Camp Levels

Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565–L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly.

Transmembrane Calcium Influx Associated with von Willebrand Factor Binding to GP Ib in the Initiation of Shear-Induced Platelet Aggregation

1993 ◽  
Vol 69 (05) ◽  
pp. 496-502 ◽  
Author(s):  
Yasuo Ikeda ◽  
Makoto Handa ◽  
Tetsuji Kamata ◽  
Koichi Kawano ◽  
Yohko Kawai ◽  
...  
Keyword(s):  
Shear Force ◽  
Calcium Influx ◽  
Fold Increase ◽  
Intracellular Camp ◽  
Recombinant Fragment ◽  
Transmembrane Flux ◽  
Irreversible Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Camp Levels

SummaryWe found that the binding of multimeric vWF to GP Ib under a shear force of 108 dynes/cm2 resulted in the transmembrane flux of Ca2+ ions with a two-to three-fold increase in their intracellular concentration ([Ca2+]i). The blockage of this event, obtained by inhibiting the vWF-GP Ib interaction, suppressed aggregation. In contrast, the blockage of vWF binding to GP IIb-IIIa, as well as the prevention of activation caused by increased intracellular cAMP levels, inhibited aggregation but had no significant effect on [Ca2+]i increase. A monomeric recombinant fragment of vWF containing the GP Ib-binding domain of the molecule (residues 445-733) prevented all effects mediated by multimeric vWF but, by itself, failed to support the increase in [Ca2+]i and aggregation. These results suggest that the binding of multimeric vWF to GP Ib initiates platelets aggregation induced by high shear stress by mediating a transmembrane flux of Ca2+ ions, perhaps through a receptor-dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of GP IIb-IIIa; the latter receptor then binds vWF and mediates irreversible aggregation.

Trabecular Meshwork Stem Cells Could Be Differentiated to Be Trabecular Meshwork Cells with Secretory Functions

2019 ◽  
Author(s):  
Ying Su ◽  
Xin Jiang ◽  
Elizabeth (Xiaomeng) Wang ◽  
Feng Wang ◽  
Ying Han
Keyword(s):  
Stem Cells ◽  
Trabecular Meshwork ◽  
Trabecular Meshwork Cells

Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (8) ◽  
Author(s):  
Sarah Giles ◽  
Uwe Stroeher ◽  
Melissa Brown
Keyword(s):  
Cyclic Nucleotide ◽  
Intracellular Camp ◽  
Camp Levels

Mechanical stretch upregulates connexin43 in human trabecular meshwork cells

2019 ◽  
Author(s):  
Nikoleta Tellios ◽  
Mary Feng ◽  
Nancy Chen ◽  
Hong Liu ◽  
Vasiliki Tellios ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Mechanical Stretch ◽  
Trabecular Meshwork Cells

Effect of dexamethasone on the expression of MMPs, adenosine A1 receptors and NFKB by human trabecular meshwork cells

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Normie Aida Mohd Nasir ◽  
Renu Agarwal ◽  
Anna Krasilnikova ◽  
Siti Hamimah Sheikh Abdul Kadir ◽  
Igor Iezhitsa
Keyword(s):  
Extracellular Matrix ◽  
Western Blot ◽  
Trabecular Meshwork ◽  
Culture Media ◽  
Smooth Muscle Actin ◽  
Adenosine A1 Receptors ◽  
Trabecular Meshwork Cells ◽  
Aqueous Humor Dynamics ◽  
Nfkb Activation ◽  
Adenosine A1

AbstractObjectivesSteroid-induced ocular hypertension and glaucoma are associated with extracellular matrix remodeling at the trabecular meshwork (TM) of the eye due to reduced secretion of matrix metalloproteinases (MMPs), a family of enzymes regulating extracellular matrix proteolysis. Several biological functions of steroids are known to involve regulation of adenosine A1 receptors (A1AR) and nuclear factor kappa B (NFKB). Since MMPs expression in TM has been shown to be regulated by A1AR as well as transcription factors, it is likely that dexamethasone-induced changes in aqueous humor dynamics involve reduced MMP and A1AR expression and reduced NFKB activation. Hence, the current study investigated the association of dexamethasone-induced reduction in MMP secretion with reduced NFKB activation and A1AR expression.MethodsHuman trabecular meshwork cells (HTMCs) were characterized by estimating myocilin and alpha smooth muscle actin expression and then were treated with dexamethasone 100 nM for 2, 5 and 7 days. The MMP secretion was estimated in culture media using Western blot. Immunocytochemistry (ICC) and ELISA were done to investigate the effect of dexamethasone on NFKB phosphorylation. A1AR expression in HTMCs was determined using Western blot and ELISA.ResultsDexamethasone caused a significant reduction in both MMP-2 and -9 expression compared to untreated group after five and seven days but not after two days of culture. Significantly reduced phosphorylated NFKB and A1AR protein levels were detected in dexamethasone treated compared to vehicle treated HTMCs after five days of culture.ConclusionsDexamethasone reduces MMP-2 and -9 secretion by HTMCs and this effect of dexamethasone is associated with reduced NFKB phosphorylation and A1AR expression.

scholarly journals Novel Template Plasmids pCyaA’-Kan and pCyaA’-Cam for Generation of Unmarked Chromosomal cyaA’ Translational Fusion to T3SS Effectors in Salmonella

2021 ◽  
Vol 9 (3) ◽  
pp. 475
Author(s):  
Paulina A. Fernández ◽  
Marcela Zabner ◽  
Jaime Ortega ◽  
Constanza Morgado ◽  
Fernando Amaya ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Eukaryotic Cells ◽  
Intracellular Camp ◽  
Gene Fusions ◽  
Secretion Systems ◽  
Translational Fusion ◽  
Flp Recombinase ◽  
Recombination System ◽  
Camp Levels

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

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