MUSCARINIC RECEPTOR SUBTYPES IN HUMAN AND RAT COLON SMOOTH-MUSCLE

Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [H-3]N-methylscopolamine ([H-3]NMS) by ligand binding studies. [H-3]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (K(D) values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P < 0.01) increase in muscarinic receptor density (B(max)) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [H-3]NMS binding by non-labelled compounds shows the following order in human colon: atropine > AF-DX 116 > pirenzepine. Whereas in rat colon the rank order obtained is atropine > pirenzepine > AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (K(i) = 1.08 +/- 0.08-mu-M) than those in human colon (K(i) = 1.74 +/- 0.02-mu-M) (P < 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P < 0.01) in human colon (IC50 = 1.69 +/- 0.37-mu-M) than in rat colon (IC50 = 3.78 +/- 0.75-mu-M). Unlike atropine and pirenzepine, the inhibition of [H-3]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (n(H) < 1, P < 0.01) suggesting the presence of more than one subtype of muscarininc receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent K(i1) value for the high affinity binding site is 0.49 +/- 0.07-mu-M for human colon smooth muscle and 0.33 +/- 0.05-mu-M for rat colon smooth muscle. The apparent K(i2) for the low affinity binding site is 8.01 +/- 1.0-mu-M for human samples and 6.07 +/- 1.1-mu-M for rat samples. These values are close enough to suggest that the first subtype of muscarinic receptor may be considered cardiac (M2) and the second subtype glandular (M3). The relative densities of the receptor subtypes are significantly different for both species. Human colon samples show the major densities of subtype M2, 22.62 +/- 1.11 fmol/mg protein, this represents 75.66 +/- 3.73% of the total receptors. Rat colon samples show the main proportion of subtype M3, 10.50 +/- 1.08 fmol/mg protein which represents 61.06 +/- 6.30% of the total receptors. This difference in muscarinic receptor subtype proportions could explain the different affinity patterns shown by pirenzepine and AF-DX 116 in human and rat colon samples.