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HEK293T Cells (hek293t + cell)

Distribution by Scientific Domains

Medical Sciences	75%

Selected Abstracts

Ethanol inhibits cold-menthol receptor TRPM8 by modulating its interaction with membrane phosphatidylinositol 4,5-bisphosphate

JOURNAL OF NEUROCHEMISTRY, Issue 1 2007
Jan Benedikt
Abstract Ethanol has opposite effects on two members of the transient receptor potential (TRP) family of ion channels: it inhibits the cold-menthol receptor TRPM8, whereas it potentiates the activity of the heat- and capsaicin-gated vanilloid receptor TRPV1. Both thermosensitive cation channels are critically regulated by the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2). The effects of this phospholipid on TRPM8 and TRPV1 are also functionally opposite: PIP2 is necessary for the activation of TRPM8 but it constitutively inhibits TRPV1. This parallel led us to investigate the possible role of PIP2 in the ethanol-induced modulation of rat TRPM8, heterologously expressed in HEK293T cells. In this study, we characterize the effects of ethanol (0.1,10%) on whole-cell currents produced by menthol and by low temperature (< 17°C). We show that the inclusion of PIP2 in the intracellular solution results in a strong reduction in the ethanol-induced inhibition of menthol-evoked responses. Conversely, intracellular dialysis with anti-PIP2 antibody or with the PIP2 scavenger, poly l -lysine, enhanced the ethanol-induced inhibition of TRPM8. A 20 min pre-incubation with wortmannin caused a modest decrease in inhibition produced by 1% ethanol, indicating that the ethanol-induced inhibition is not mediated by lipid kinases. These findings suggest that ethanol inhibits TRPM8 by weakening the PIP2,TRPM8 channel interaction; a similar mechanism may contribute to the ethanol-mediated modulation of some other PIP2 -sensitive TRP channels. [source]

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects

BRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2007
D G Johns
Background and purpose: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified ,CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator ,CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. Experimental approach: Human recombinant GPR55 was expressed in HEK293T cells and specific GTP,S activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. Key results: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTP,S activity (EC50 approximately 2 nM), whereas the CB1 and CB2 -selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20±2%, KO 26±5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 ,M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. Conclusions: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents. British Journal of Pharmacology (2007) 152, 825,831; doi:10.1038/sj.bjp.0707419; published online 20 August 2007 [source]

LIM domain-containing adaptor, leupaxin, localizes in focal adhesion and suppresses the integrin-induced tyrosine phosphorylation of paxillin

CANCER SCIENCE, Issue 2 2010
Toshiyuki Tanaka
Focal adhesion (FA) consists of multiple cellular proteins including paxillin and serves as a center for adhesion-mediated signaling. The assembly and disassembly of FAs is regulated by locally produced intracellular signals, and tyrosine phosphorylation of paxillin has been implicated in this process. A Lin-11 Isl-1 Mec-3 (LIM) domain-containing adaptor protein, leupaxin, a member of the paxillin family, is expressed in leukocytes as well as in certain cancer cells, and shares overall structural characteristics with paxillin. However, it remains unknown whether leupaxin and paxillin cooperate with or antagonize each other in integrin signaling. Here we show that leupaxin potently represses the tyrosine phosphorylation of paxillin. When expressed in mouse thymoma BW5147 cells bound to ICAM-1, leupaxin accumulated in FA-like patches in the cell periphery. When expressed in NIH3T3 and HEK293T cells, leupaxin localized to FAs upon cell adhesion to fibronectin and strongly suppressed the integrin-induced tyrosine phosphorylation of paxillin. In integrin-stimulated HEK293T cells, leupaxin's LIM3 domain appeared essential for selective FA localization and the suppression of paxillin tyrosine phosphorylation. Leupaxin's LD3 motif, which is critical for stable association with FAK, was dispensable for leupaxin's suppressive ability. In addition, leupaxin reduced the spreading of NIH3T3 cells on fibronectin, which required both the LD3 motif and LIM3 domain. When expressed in human leukocytic K562 cells, leupaxin significantly suppressed integrin ,5,1-mediated cell adhesion to fibronectin and the tyrosine phosphorylation of paxillin. These findings indicate that leupaxin functions as a paxillin counterpart that potently suppresses the tyrosine phosphorylation of paxillin during integrin signaling. (Cancer Sci 2009) [source]