Specific bindings of [3H](+)PN200-110 and [125I]?-conotoxin to crude membranes from differentiated NG108-15 cells

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Abstract
The characteristics of the specific bindings of [3H](+)PN200-110 (PN: L-type Ca channel antagonist) and [125I]ω-conotoxin G VI A (ω-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt2cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated45Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt2cAMP (unpublished results). The specific binding of [3H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [3H](+)PN. Scatchard analysis showed that the specific binding of [3H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt2cAMP, but that the apparent Kd value for the specific binding of [3H](+)PN was not influenced by treatment with Bt2cAMP. The specific binding of [3H](+)PN to crude membranes from Bt2cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(−)PN≧Bay K 8644≫diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [3H(+)PN. On the other hand, [125I]ω-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated45Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [125I]ω-CgTX on Bt2cAMP-treated or untreated NG108-15 cells.