EFFECTS OF METAL-CATIONS ON [H-3] ALPHA,BETA-METHYLENE ATP BINDING INRAT VAS-DEFERENS

In this study we have examined the effect of metal cations (as their c hloride salts) on the binding of [H-3]alpha,beta-methylene ATP ([H-3]a lpha beta meATP) to rat vas deferens membranes using a vacuum filtrati on receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did n ot affect total binding of 1 nM [H-3]alpha beta meATP, several divalen t and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl3 and AlCl3 (0.1 to 100 mu M), produced the greates t increases in total binding of [H-3]alpha beta meATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 mu M and 1-10 mM, increased total binding of [H-3]alpha beta meATP to rat v as deferens by between 87% and 215% while having no effect on either f ilter binding or non specific binding. The following pEC(50) values fo r potentiating binding of the radioligand were obtained: ZnCl2 (5.44), MnCl2 (4.52), CaCl2 (4.17), CoCl2 (4.06), MgCl2 (3.67) and BaCl2 (3.1 0). Both EDTA and EGTA (0.01-1 mM) inhibited the binding of the radiol igand. The effects of ZnCl2, CaCl2 and MgCl2 were examined in saturati on studies. In the absence of added divalent cations, [H-3]alpha beta meATP labelled both high (pK(d) = 9.15) and low (pK(d) = 7.06) affinit y binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl2 (pK(d) = 9.56) and by 30 mu M ZnCl2 (pK(d) = 9.46) but not by 3 mM MgCl2. The B-max of the low affinity s ite for [H-3]alpha beta meATP was increased (approximately 4 fold) by both 3 mM MgCl2 and 30 mu M ZnCl2 but not by 3 mM CaCl2. The selective effect of CaCl2 on the high affinity binding sites enabled these site s to be labelled in the presence of 3 mM CaCl2 using a low concentrati on of [H-3]alpha beta meATP (1 nM); the sites exhibited the binding ch aracteristics expected of the P-2x purinoceptor. The selective effect of MgCl2 on the low affinity binding sites enabled these sites to be l abelled in the presence of 3 mM MgCl2 and using a high concentration o f [H-3]alpha beta meATP (100 nM). A comparison of the binding characte ristics of the high and low affinity sites for [H-3]alpha beta meATP r evealed several other differences, in addition to their cation selecti vity. First, the adenine analogues ADP, alpha beta meATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for t he high affinity [H-3]alpha beta meATP binding sites than for the low affinity binding sites. Secondly, GTP-gamma-S and pyrophosphate were s elective ligands for the low affinity [H-3]alpha beta meATP binding si tes possessing approximately 43 and 1995 fold, respectively, higher pI C(50) values at the low affinity sites than at the high affinity sites . Finally, treatment of the membranes with 0.01-1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of th e low affinity sites suggest that they do not equate with functional P -2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [H-3]alpha beta meATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity. In conclusion the-present study has demonstrated that cations have marked effect on the binding of [H-3]alpha beta meATP in rat vas deferens. O f particular interest was the ability of CaCl2 to increase the affinit y of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl2 to increase the B-max of the low affinity binding sites enabled these sites to be sel ectively labeled.