Effects of substance P on the binding of ligands to nicotinic acetylcholine receptors

The effect of substance P on the binding of many ligands that interact with the nicotinic acetylcholine receptor was examined in membrane preparations of Torpedo electroplaque and BC3H-1 cells and in solubilized membranes of rat, chick, and goldfish brain. In the absence of carbamylcholine, the affinity of [3H]phencyclidine for the high affinity local anesthetic binding site on Torpedo membranes was increased by substance P with an EC50 of approximately 5 microM. In the presence of carbamylcholine, which itself increases [3H]phencyclidine binding affinity, substance P caused a decrease in the affinity of [3H]phencyclidine. The concentration dependence of the inhibition, however, was inconsistent with a competitive interaction, since the apparent Hill coefficient was significantly less than one. We conclude from these results that substance P does not directly interact with the high affinity local anesthetic binding site on the nicotinic receptor of Torpedo membranes. Substance P also does not appear to interact directly with the agonist binding site since the peptide had no significant effect on [3H]acetylcholine binding to Torpedo membranes. Substance P inhibited [125I]alpha-bungarotoxin binding to both native and Triton X-100 solubilized Torpedo membranes, although the IC50 was 8-fold higher for the solubilized preparation (12 versus 93 microM). We interpret this inhibition in solubilized membranes as evidence that the peptide may interact directly with a binding site on the nicotinic acetylcholine receptor. Substance P also decreased the initial rate of [125I]alpha-bungarotoxin to membranes prepared from BC3H-1 cells (IC50 = 108 microM) and to solubilized membranes from rat, chick, and goldfish brain. In the brain membranes, however, the peptide did not completely inhibit binding; at the highest concentration examined (100 microM), the maximum inhibition observed was 60%. Consistent with the results for [3H]acetylcholine binding to Torpedo membranes, the peptide had no effect on the binding of the cholinergic agonist [3H](-)nicotine to these tissue preparations. These data suggest that substance P may have a general modulatory action on a subclass of nicotinic receptors that include muscle-type, ganglionic-type, and a putative subpopulation of central nervous system receptors.