TY - JOUR
T1 - Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells
AU - Yang, Chuen Mao
AU - Tsai, Yih Jeng
AU - Pan, Shiow Lin
AU - Tsai, Chuen Tao
AU - Wu, Wen Bin
AU - Chiu, Chi Tso
AU - Luo, Shu Fen
AU - Ou, Jonathan T.
PY - 1997
Y1 - 1997
N2 - Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (U?) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPγS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPγS > ATP = 2-chloro ATP (Cl-ATP) >> α,β-methylene ATP (α,β-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P(2X)- and P(2Y)-purinoceptors, such as N6-cyclopentyl adenosine, AMP, α,β-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPγS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P(2U)-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPγS were Shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the UP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P(2U)-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.
AB - Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (U?) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPγS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPγS > ATP = 2-chloro ATP (Cl-ATP) >> α,β-methylene ATP (α,β-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P(2X)- and P(2Y)-purinoceptors, such as N6-cyclopentyl adenosine, AMP, α,β-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPγS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P(2U)-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPγS were Shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the UP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P(2U)-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.
KW - Inositol phosphate accumulation
KW - Kidney cells
KW - Madin-Darby canine
KW - P(2U)-purinoceptor
KW - Pertussis toxin
KW - Phosphoinositide
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U2 - 10.1007/PL00005015
DO - 10.1007/PL00005015
M3 - Article
C2 - 9228183
AN - SCOPUS:0030910189
SN - 0028-1298
VL - 356
SP - 1
EP - 7
JO - Naunyn-Schmiedeberg's Archives of Pharmacology
JF - Naunyn-Schmiedeberg's Archives of Pharmacology
IS - 1
ER -