Pm-purinoceptor-stimulated phosphoinositide hydrolysis in madindarby canine kidney cells

Extracellular nucleotides, acting through P₂-purinoceptors, have been implicated in the regulation of ion transporting 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 (IP) 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). The selective agonists for P₁-, P_2X- and P_2Y1-purinoceptors, such as N⁶-cyclopentyl adenosine, AMP, α,β-MeATP, 2MeSATP, had little effect on this response. Stimulation of MDCK cells with maximally effective concentration of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPγS induced cross-desensitization of the IP accumulation response, suggesting that ATP and UTP acted upon a common nucleotide receptor, that was P_2Y2-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, but asymptotic maxima were not reached, indicating that either MgATP^2- or CaATP^2- was not the active agonist, which was likely to be ATP⁴. 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 IP accumulation, indicating that a PTX-sensitive G protein was involved in this IP response. Thus, these results conclude that ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P_2Y2-purinoceptor coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ion such as ATP^4- seems to be the preferential agonist form for the purinoceptor on MDCK cells.