Intracellular calcium mobilization on stimulation of the muscarinic cholinergic receptor in chick limb bud cells

Abstract

Cell suspensions of chick limb buds (stage 23/24) were loaded with the fluorescent Ca²⁺ chelator chlorotetracycline. Fluorescence was monitored in a spectrofluorometer. Stimulation with acetylcholine induced a fluorescence decrease, indicating intracellular Ca²⁺ mobilization. The fluorescence decrease triggered by acetylcholine was inhibited by muscarinic but not by nicotinic antagonists, indicating that a muscarinic acetylcholine receptor is involved. The muscarinic receptor in the chick limb bud has a characteristic pharmacological profile: acetylcholine, carbachol and acetyl-β-methylcholine functioned as full agonists triggering maximal fluorescence decrease. Bethanechol was less effective, producing only one-third of the maximum response. Pilocarpine and oxotremorine, two classical agonists in other systems, were ineffective and functioned as antagonists. In the chick limb bud, cholinesterase, choline acetyltransferase and the presence of a muscarinic receptor have been demonstrated in previous studies. The present experiments show that stimulation of the embryonic muscarinic receptor leads to intracellular Ca²⁺ mobilization.

Keywords: Calcium mobilization; Chlorotetracycline; Limb bud; Morphogenesis; Muscarinic cholinergic receptor.