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Human Colon Cancer Cell Proliferation Mediated by the M₃ Muscarinic Cholinergic Receptor¹

Division of Oncologic Gastroenterology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [H. F., D. D., W-L. Y., Y. X.], and Digestive Diseases Branch, NIH, Bethesda, Maryland 20892 [R. T. J.]

We have demonstrated previously cell surface receptors for gastrointestinal peptides on 10 human colon cancer cell lines. Because most of the cells studied 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 specifically, the effect on cell proliferation. We used radiolabeled ligand binding, PCR, calcium mobilization, and cellular proliferation studies. The present study demonstrates a muscarinic cholinergic receptor having two classes of binding site for carbamylcholine. Analysis demonstrated 2499 ± 153 binding sites/cell, of which 75% had a high affinity for carbamylcholine (K_d 55 µM), and 25% had a low affinity (K_d 0.33 mM). N-Methylscopolamine, a receptor 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 chief cells in physiological conditions. Specific cholinergic receptor antagonists inhibit binding in the following order of potency: N-methylscopolamine > 4-DAMP >> pirenzipine > AF-DX116. This order of potency pharmacologically classifies the receptor as an M₃ subtype. Receptor expression, studied by reverse transcription-PCR, correlates with the binding data. Specifically, cell lines that exhibit binding, abundantly expressed the M₃ receptor subtype, whereas cell lines that do not exhibit binding for muscarinic cholinergic agonists did not abundantly express the M₃ receptor. Agonist activation of the M₃ receptor on these cells resulted in intracellular calcium mobilization. The dose-response curve of calcium mobilization suggests that there are spare receptors on these cells. Signal transduction can be inhibited by receptor antagonists in the same order of potency in which the binding is inhibited. Exogenous agonist added to the cells in culture induces significant cell proliferation. These results demonstrate a muscarinic cholinergic receptor of the M₃ 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, and that there may be enhanced intracellular signal activation in response to receptor binding.

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