Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells

Chuen Mao Yang, Yih Jeng Tsai, Shiow Lin Pan, Chuen Tao Tsai, Wen Bin Wu, Chi Tso Chiu, Shu Fen Luo, Jonathan T. Ou

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1-7
Number of pages7
JournalNaunyn-Schmiedeberg's Archives of Pharmacology
Volume356
Issue number1
DOIs
Publication statusPublished - 1997
Externally publishedYes

Keywords

  • Inositol phosphate accumulation
  • Kidney cells
  • Madin-Darby canine
  • P(2U)-purinoceptor
  • Pertussis toxin
  • Phosphoinositide

ASJC Scopus subject areas

  • Pharmacology

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