Characterization of P2X₃, P2Y₁ and P2Y₄ receptors in cultured HEK293‐hP2X₃ cells and their inhibition by ethanol and trichloroethanol

Abstract

Membrane currents and changes in the intracellular Ca ] i was observed after the slow superfusion of ATP, ADPb-S and UTP; a,b-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to a,b-meATP is due to P2X 3 receptor activation, while the ATP-induced rise in [Ca 2+ ] i is evoked by P2Y 1 and P2Y 4 receptor activation. TCE depressed the a,b-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca 2+ ] i was much less sensitive to the inhibitory effect of TCE than the current response to a,b-meATP. The present study indicates that in HEK293-hP2X 3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X 3 receptors and, in addition, moderately interferes with G protein-coupled P2Y 1 and P2Y 4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.

Regulation of Human Recombinant P2X3Receptors by Ecto-Protein Kinase C

“…Because GDP-␤-S is known to inhibit G-protein-mediated second-messenger mechanisms (Sternweis and Pang, 1990) of, for example, P2Y receptors (Ralevic and Burnstock, 1998), it has been suggested that endogenous P2Y receptors of HEK293 cells may negatively interact with the exogenous hP2X 3 receptor (Fischer et al, 2003). In the present study, UTP may exert both a P2Y 4 receptor-mediated depression and a hitherto unknown facilitation of P2X 3 receptors.…”

“…Potentiation by UTP of the ␣,␤-meATP current amplitude in HEK293-hP2X 3 cells and interaction with protein kinase C inhibitors Although in HEK293-hP2X 3 cells, ATP, ␣,␤-meATP, and UTP caused, at a holding potential of Ϫ60 mV, inward currents, the threshold concentrations of ATP and ␣,␤-meATP were Ͼ0.3 M, whereas the threshold concentration of UTP was considerably higher (Ͼ10 M) (Fischer et al, 2003). In accordance with these results, UTP (3 M) did not evoke a current response by itself and also failed to alter the effect of ␣,␤-meATP (3 M; 1 s superfusion time) (Fig.…”

“…HEK293 cells have been shown to possess a range of endogenous P2Y receptors that preferentially react either to ADP (P2Y 1 , P2Y 13 ) or UTP (P2Y 4 ) (Fischer et al, 2003). Because GDP-␤-S is known to inhibit G-protein-mediated second-messenger mechanisms (Sternweis and Pang, 1990) of, for example, P2Y receptors (Ralevic and Burnstock, 1998), it has been suggested that endogenous P2Y receptors of HEK293 cells may negatively interact with the exogenous hP2X 3 receptor (Fischer et al, 2003).…”

“…Methods of maintenance of HEK293 cells and stable transfection of them with hP2X 3 receptor cDNA have been described previously (Fischer et al, 2003). The cDNA of the wild-type (WT) P2X 3 receptor (Dr. J. N. Wood, University College London, London, UK) was cloned into the bicistronic vector pIRES2/EGFP (Clontech, Heidelberg, Germany), containing the cDNA of enhanced green fluorescence protein to enable the identification of efficiently transfected cells under a fluorescence microscope.…”

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

“…HEK293 cells express metabotropic P2Y receptors capable of increasing the concentration of intracellular Ca 2ϩ ([Ca 2ϩ ] i ) by mobilizing internal Ca 2ϩ stores in a GTP-dependent manner (76,77). We used a HEK293 cell line with stable expression of the genetically encoded Ca 2ϩ sensor, GCaMP5G (78), to determine whether activation of P2Y receptors by ATP (6 mM) is an unintended consequence of using high agonist concentrations to measure Pf %.…”