MUSCARINIC RECEPTORS IN CANINE COLONIC CIRCULAR SMOOTH-MUSCLE .1. COEXISTENCE OF M2 AND M3 SUBTYPES

The parasympathetic neurotransmitter acetylcholine, acting postsynapti cally at the smooth muscle muscarinic receptor, is a principle determi nant of colonic motility. In order to elucidate the receptor signal-tr ansduction events responsible for muscarinic receptor-induced contract ion of colonic circular smooth muscle, we present here and in the acco mpanying work studies designed to characterize the muscarinic receptor s present in colon and to determine their biochemical coupling. Muscar inic receptor subtypes in canine colonic circular smooth muscle were c haracterized using radioligand binding techniques. The nonselective mu scarinic receptor antagonist radioligand [H-3]quinuclidinyl benzilate ([H-3]QNB) binds rapidly and reversibly to a single class of saturable sites in colon circular smooth muscle membranes, with an affinity (K( D)) for the antagonist radioligand of 79.8 +/- 12.6 pM and a density o f 123.3 +/- 18.7 fmol/mg of protein. Experiments using membranes prepa red from isolated cells purified from the circular smooth muscle layer of canine colon (K(D) = 102.4 +/- 13.5 pM) confirm the smooth muscle origin of the binding and yield a receptor density of 124,340 receptor s/cell. The order of potencies of selective muscarinic receptor antago nists in competition with [H-3]QNB for binding to colonic receptors is 4-diphenylacetoxy-N-methylpiperidine methobromide > methoctramine > A F-DX 116 > pirenzepine. Unlike other antagonists tested, pirenzepine c ompetition of [H-3]QNB binding is biphasic. The high and low affinitie s deduced from nonlinear fit of the binding data in colon correlate ve ry well with affinities determined for pirenzepine in mixtures of both submandibular gland (M3) and atrium (M2), indicating the presence of two muscarinic receptor subtypes (82% M2, 18% M3) in colon circular sm ooth muscle. The muscarinic agonist carbachol binds to both high and l ow affinity sites in colon, and addition of guanine nucleotide (100-mu -M GTP-gamma-S) shifts the agonist competition curve to the right, wit hout eliminating high affinity binding sites. Agonist competition stud ies with a known ratio of M2 and M3 receptors, obtained by mixing pure M2 and M3 populations, predict the result obtained in colon. CDNA pro bes specific for each of the muscarinic receptors m1 through m4 were h ybridized to colon RNA in a Northern blot analysis. Only m2 and m3 pro bes hybridized to colon RNA, suggesting the presence of both M2 and M3 receptors. Our data demonstrate that the colon circular smooth muscle contains muscarinic receptors of both the M2 and M3 subtypes, which m ay be coupled to disparate signal transduction pathways important in t he physiological actions of acetylcholine in this tissue.