Mixed Competitive and Allosteric Antagonism by Gallamine of Muscarinic Receptor‐Mediated Second Messenger Responses in N1E‐115 Neuroblastoma Cells

Abstract: The antagonistic effects of gallamine on muscarinic receptor‐linked responses were investigated in N1E‐115 neuroblastoma cells. M₁ muscarinic receptor‐mediated phos‐phoinositide hydrolysis induced by carbamylcholine was antagonized by gallamine, with a K_i value of 33 μM. By comparison, gallamine was four‐ to fivefold less potent in blocking noncardiac M₂ muscarinic receptor‐mediated inhibition of cyclic AMP formation, with a K_i value of 144 μM. The resulting Arunlakshana‐Schild plots of the antagonism of both responses by gallamine were linear and exhibited slopes not differing from 1, a result indicative of a competitive mechanism. To elucidate further the nature of gallamine's inhibitory actions, experiments were performed where the effects of gallamine in combination with the known competitive muscarinic antagonist, N‐methylscopolamine (NMS), were studied. In the presence of both antagonists, a supraadditive shift in the carbamylcholine dose‐response curve was demonstrated for the two responses, a result suggestive of an allosteric mode of interaction between gallamine and NMS binding sites. Confirmation that gallamine allosterically modifies the muscarinic receptor was provided by radioligand binding studies. Gallamine competition curves with either [N‐methyl‐³H]scopolamime methyl chloride ([³H]NMS) or [N‐methyl‐³H]quinuclidinyl benzilate methyl chloride ([³H]NMeQNB) were unusually shallow. Furthermore, gallamine decelerated the rate of dissociation of receptor‐bound [³H]NMS > [³HJNMeQNB in a dose‐dependent manner. The present study demonstrates that whereas gallamine antagonizes carbamylcholine‐mediated responses in N1E‐115 cells in a competitive manner, an allosteric component of its action is revealed in the presence of muscarinic antagonists such as NMS.