Human colon cancer cell proliferation mediated by the M-3 muscarinic cholinergic receptor

We have demonstrated previously cell surface receptors for gastrointestinal peptides on 10 human colon cancer cell lines, Because most of the cells st udied bind muscarinic cholinergic agonists, we undertook the determination of the cholinergic receptor subtype expressed by human colon cancer cells, as well as the biological function of these receptors, and more specificall y, the effect on cell proliferation. me used radiolabeled ligand binding, P CR, calcium mobilization, and cellular proliferation studies. The present s tudy demonstrates a muscarinic cholinergic receptor having two classes of b inding site for carbamylcholine. Analysis demonstrated 2499 +/- 153 binding sites/cell, of which 75% had a high affinity for carbamylcholine (K-d 55 m u M), and 25% had a low affinity (K-d 0.33 mM). N-Methylscopolamine, a rece ptor antagonist, recognized only one binding site having high affinity (K-d 0.20 nM). The number of muscarinic cholinergic binding sites/cell found on colon cancer cells is 50% of the number of receptors found on guinea pig c hief cells in physiological conditions. Specific cholinergic receptor antag onists inhibit binding in the following order of potency: N-methylscopolami ne > 4-DAMP much greater than pirenzipine > AFDX116, This order of potency pharmacologically classifies the receptor as an M-3 subtype, Receptor expre ssion, studied by reverse transcription-PCR, correlates with the binding da ta. Specifically, cell lines that exhibit binding, abundantly expressed the M-3 receptor subtype, whereas cell lines that do not exhibit binding for m uscarinic cholinergic agonists did not abundantly express the M, receptor. Agonist activation of the M, receptor on these cells resulted in intracellu lar calcium mobilization, The dose-response curve of calcium mobilization s uggests that there are spare receptors on these cells. Signal transduction can be inhibited by receptor antagonists in the same order of potency in wh ich the binding is inhibited. Exogenous agonist added to the cells in cultu re induces significant cell proliferation. These results demonstrate a musc arinic cholinergic receptor of the M-3 subtype on human colon cancer cells. This receptor Induces intracellular calcium mobilization and mediates cell proliferation. The data suggest that there are spare receptors present, an d that there may be enhanced intracellular signal activation in response to receptor binding.