Analysis by
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis of
cholinergic receptor protein affinity labeled with the
muscarinic antagonist [3H]
propylbenzilylcholine mustard revealed a major
polypeptide with molecular weight of 80,000-83,000 in both
acinar carcinoma and normal acinar cells of rat pancreas.
Muscarinic receptor protein is therefore conserved in
pancreatic acinar carcinoma. A small but significant difference was detected in the affinity of
carcinoma cell receptors (Kd approximately 0.6 nM) and normal cell receptors (Kd approximately 0.3 nM) for reversible binding of the
muscarinic antagonist drug,
N-methylscopolamine. In addition,
carcinoma cell
muscarinic receptors displayed homogeneous binding of the agonist drugs
carbamylcholine (Kd approximately 31 microM) and
oxotremorine (Kd approximately 4 microM), whereas normal cell receptors demonstrated heterogeneous binding, with a minor receptor population showing high affinity binding for
carbamylcholine (Kd approximately 3 microM) and
oxotremorine (Kd approximately 160 nM), and a major population showing low affinity binding for
carbamylcholine (Kd approximately 110 microM) and
oxotremorine (Kd approximately 18 microM). Both
carcinoma and normal cells exhibited concentration-dependent
carbamylcholine-stimulated increases in cytosolic free Ca2+, as measured by 45Ca2+ outflux assay and intracellular
quin 2 fluorescence. However,
carcinoma cells were observed to be more sensitive to Ca2+ mobilizing actions of submaximal
carbamylcholine concentrations, demonstrating 50% maximal stimulation of intracellular Ca2+ release at a
carbamylcholine concentration (approximately 0.4 microM) approximately one order of magnitude below that seen for normal cells. These results indicate altered
muscarinic receptor coupling to intracellular Ca2+ release in
acinar carcinoma cells, which manifests as a single activated receptor state for agonist binding, and increased sensitivity of Ca2+ release in response to
muscarinic receptor stimulation.