MUSCARINIC RESPONSES AND BINDING IN A MURINE NEURO-BLASTOMA CLONE (N1E-115) - MEDIATION OF SEPARATE RESPONSES BY HIGH-AFFINITY AND LOW AFFINITY AGONIST-RECEPTOR CONFORMATIONS

  • 1 January 1985
    • journal article
    • research article
    • Vol. 27  (2) , 223-235
Abstract
Murine neuroblastoma cells (clone N1E-115) possess muscarinic receptors that mediate multiple responses, including the elevation of cGMP levels and the inhibition of receptor-mediated increases in cAMP. Evidence is presented showing that 2 muscarinic agonist-receptor conformations in N1E-115 cells each separately mediate a cyclic nucleotide response. Pirenzepine inhibited the [3H]cGMP response to carbachol with a Kd value of approximately 6 nM, whereas it inhibited the ability of carbachol to reduce prostaglandin E1-mediated elevations in [3H]cAMP levels with a Kd value of 93 nM, thus differentiating between 2 classes of receptors involved in these responses. Muscarinic agonists (10) were studied for their ability to mediate the 2 cyclic nucleotide responses. Six were as effective as acetylcholine in the reduction of [3H]cAMP levels, but only 2 were as effective as acetylcholine in elevating [3H]cGMP levels. Agonists (4) (arecoline, pilocarpine, oxotremorine and McN-A343) were ineffective in increasing [3H]cGMP levels only 18% as well as acetylcholine, behaved as competitive antagonists in this response to carbachol. These partial agonists, in contrast to carbachol, bound to only 1 class of muscarinic sites in N1E-115 cells with equilibrium dissociation constants determined by competition binding assays which agreed well with their respective EC50 [concentration giving 50% effectiveness] values for their effect on [3H]cAMP levels. The equilibrium Kd for the partial agonists determined by their inhibition of carbachol in the [3H]cGMP response also agreed well with their respective EC50 values for mediating the [3H]cAMP response. The partial agonists bound to the same receptors at which carbachol mediated [3H]cGMP formation, but with Kd values about the same as their respective EC50 values for inhibition of prostaglandin E1-mediated [3H]cAMP increases. The full agonists acetylcholine and methacholine, like carbachol, bound to 2 sites in N1E-115 cells. For the 6 agonists able to stimulate both responses at least to some degree, the ratio of their potencies at each response correlated with their respective efficacies at each response but with much more dependence in the [3H]cGMP response. The low affinity agonist-receptor conformation, inducible by full agonists, activates the effector for the cGMP response and its formatoin is inhibited by pirenzepine with high potency; and the high affinity agonist-receptor conformation, inducible by both full and partial agonists, activates the effector for the cAMP reduction and is inhibited by pirenzepine with lower potency.