Effects of metal cations on [3H]alpha,beta-methylene ATP binding in rat vas deferens

Abstract

In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [3H]alpha,beta-methylene ATP ([3H]alpha beta meATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [3H]alpha beta meATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl3 and AlCl3 (0.1 to 100 microM), produced the greatest increases in total binding of [3H]alpha beta meATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 microM and 1-10 mM, increased total binding of [3H]alpha beta meATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC50 values for potentiating binding of the radioligand were obtained: ZnCl2 (5.44), MnCl2 (4.52), CaCl2 (4.17), CoCl2 (4.06), MgCl2 (3.67) and BaCl2 (3.10). Both EDTA and EGTA (0.01-1 mM) inhibited the binding of the radioligand. The effects of ZnCl2, CaCl2 and MgCl2 were examined in saturation studies. In the absence of added divalent cations, [3H]alpha beta meATP labelled both high (pKd = 9.15) and low (pKd = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl2 (pKd = 9.56) and by 30 microM ZnCl2 (pKd = 9.46) but not by 3 mM MgCl2. The Bmax of the low affinity site for [3H]alpha beta meATP was increased (approximately 4 fold) by both 3 mM MgCl2 and 30 microM ZnCl2 but not by 3 mM CaCl2. The selective effect of CaCl2 on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl2 using a low concentration of [3H]alpha beta meATP (1 nM); the sites exhibited the binding characteristics expected of the P2x purinoceptor. The selective effect of MgCl2 on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl2 and using a high concentration of [3H]alpha beta meATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [3H]alpha beta meATP revealed several other differences, in addition to their cation selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)