Introduction: Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting. Materials and Methods: Intracellular calcium concentration ([Ca2+]i) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[3H]inositol- (0.5 µCi/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used. Results: Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca2+]i, cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the Gi inhibitor pertussis toxin. Agonists potently stimulated migration of T24 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration. Conclusions: G protein-coupled receptors potently stimulated cell migration in T24 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer.
Membrane-permeable 8-(4-chlorophenylthio)-2'-O-methyl cyclic AMP (8-pCPT-2'-O-Me-cAMP) has been shown to specifically activate cAMP-regulated Epac proteins, without direct effects on protein kinase A and protein kinase G. During isometric tension measurements in thoracic aortic rings from Wistar rats, we observed that 8-pCPT-2'-O-Me-cAMP selectively induced a rightward shift of the concentration response curve for the thromboxane mimetic U46619, without altering the contractile response to noradrenaline. We hypothesised that 8-pCPT-2'-O-Me-cAMP and similar compounds may function as direct thromboxane receptor antagonists. Indeed, in addition to 8-pCPT-2'-O- Me-cAMP, also 8-pCPT-cAMP, 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-CPT-cAMPS) and 8-CPT-adenosine, but not 8-Bromo-2'-O-Me-cAMP, induced rightward shifts of the contractile response to U46619. Likewise, 8-pCPT-2'-O- Me-cAMP and Rp-8-CPT-cAMPS, but not 8-Bromo-2'-O-Me-cAMP, specifically reduced U46619-induced aggregation of human platelets. In addition, 8-pCPT-2'-O-Me-cAMP and Rp-8-CPT-cAMPS completely reversed U46619-induced reduction of intercellular adhesion molecule-1 expression and migration of human coronary artery endothelial cells. Most important, the cAMP analogs that reduced the contractile response to U46619 also concentration-dependently inhibited binding of the thromboxane receptor radioligand [5,6-3H]SQ29548 to human platelets. We conclude that 8-pCPT-conjugated cAMP analogs exert competitive thromboxane receptor antagonistic properties.
Dynamic transrectal ultrasound is a reliable and reasonable method to identify extravasation after RRP. Furthermore radiation exposure (on average 60 cGy/cm(2)/cystoradiogram) can be avoided by replacing the cystoradiogram with dTRUS slips with the dTRUS.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.