Muscarinic receptor subtypes in bovine adrenal medulla

Muscarinic receptors in bovine adrenal medullary microsomes were characterized by radioligand binding assay, using l-[3H]quinuclidinyl benzilate (QNB), a muscarinic antagonist. Specific [3H]QNB binding to microsomes was rapid, reversible, saturable and of high affinity. Saturation experiments revealed a single class of binding sites for the radioligand with a maximum number of binding sites and an apparent dissociation constant of 162.6 fmoles/mg protein and 40.3 pM respectively. According to computer-assisted nonlinear regression analysis, however, drug/[3H]QNB competition curves indicated the presence of at least two affinity sites for muscarinic agonists (acetylcholine, carbamylcholine, oxotremorine), with a high (K1) and a low (K2) affinity (e.g. K1 = 664.8 nM and K2 = 36.5 microM for acetylcholine). The two affinity sites for acetylcholine showed only minimal regulation by magnesium and guanosine 5'-triphosphate. Furthermore, the presence of two affinity sites was suggested for the antagonists pirenzepine and gallamine, but not for atropine and pilocarpine. The K1 and K2 values for pirenzepine were 23.7 and 429 nM, respectively, with 54.5% of total sites having a high affinity. These results indicate that at least two distinct subtypes of muscarinic receptors exist in the bovine adrenal medulla and that they are distinguished by their relative binding affinity for muscarinic agonists and antagonists. The receptors are predominantly composed of the affinity state termed M1, as described for the receptors of sympathetic ganglia.