BINDING-KINETICS OF ATP(GAMMA)S-35 ON CULTURED PRIMARY TRACHEAL SURFACE EPITHELIAL-CELLS

Extracellular ATP can stimulate mucin release from primary hamster tra cheal surface epithelial (HTSE) cells via a P2 purinoceptor-mediated m echanism, based on agonist potency studies of mucin release (Br. J. Ph armacol. 1991; 103:1053-1056). In the present study, we examined the k inetics of ATP binding to the surface of intact HTSE cells at 4-degree s-C using ATP S-gamma(35) as a radioligand. We found that ATP S-gamma( 35) bound to HTSE cells in a saturable, reversible manner, reaching an equilibrium at about 30 min. Scatchard analysis of equilibrium bindin g suggested the presence of two binding sites with K(d) values of 0.47 and 9.4 muM. Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block ATP S-gamma(35) revealed a rank order of ATP > ADP > alpha,beta-methylene ATP > 2-methylthio ATP great er-than-or-equal-to beta,GAMMA-methylene ATP. Neither AMP nor adenosin e could inhibit the ATP S-gamma(35) binding. A comparison between the ability of nucleotides to compete with ATP S-gamma(35) binding and the ir ability to induce mucin release revealed a rather poor correlation (r2 = 0.67) with all of the above nucleotides but a good correlation ( r2 = 0.96) without 2-methylthio ATP, indicating the presence of hetero genous ATP binding sites on the HTSE cell surface. UTP, a pyrimidine n ucleotide, which is almost equipotent with ATP in its ability to stimu late mucin release, was much less potent than ATP in its ability to di splace the ATP S-gamma(35) binding in these HTSE cells.