PURINOCEPTOR-STIMULATED PHOSPHOINOSITIDE HYDROLYSIS IN MADIN-DARBY CANINE KIDNEY (MDCK) CELLS

Extracellular nucleotides, acting through P-2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments we re conducted to characterize the P-2-purinoceptor subtype on MDCK cell s responsible for stimulating inositol phosphate (IP) accumulation usi ng a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Hense leit solution (KHS), ATP, UTP, and ATP gamma S caused an increase in I P accumulation as a function of concentration with comparable kinetics . The order of potency for the nucleotide analogues was UTP = ATP gamm a S > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,b eta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P-1-, P-2X- and P-2Y-purinoceptors, such as N-6-cyclopentyl adenosine, AMP, alpha,beta-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 ATP gamma S induced cr oss-desensitization of the IP response, suggesting that ATP and UTP ac t upon a common nucleotide receptor, i.e. a P-2U-purinoceptor. In Ca2- and Mg2+-containing KHS, the concentration-response curves of ATP, U TP, and ATP gamma S were shifted to the right of those obtained in Ca2 +- and Mg2+-free buffer, and asymptotic maxima were not reached, indic ating that ATP(4-) and not MgATP(2-) or CaATP(2-) was the active agoni st. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited AT P- and UTP-induced IP accumulation in a concentration-dependent fashio n but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. I n conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells ap pears to be mediated through the activation of P-2U-purinoceptors coup led to a G protein that is partially sensitive to PTX. A form of nucle otide uncomplexed with divalent ions such as ATP(4-) seems to be the p referential agonist form for the purinoceptors on MDCK cells.