Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10–14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific α-actin. mRNA encoding muscarinic receptor subtypes M1–M5 was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. Basal [Ca2+]i, which was 135 ± 22 nM, increased transiently to 543 ± 29 nM in response to 10 μM ACh in CM cells ( n = 8). This response was decreased <95% by 0.01 μM 4-diphenylacetoxy- N-methylpiperidine, a M1/M3-selective antagonist, whereas 0.1 μM methoctramine, a M2/M4-selective antagonist, and 0.1 μM pirenzepine, a M1-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca2+]i mediated primarily by the M3 receptor and involving release of Ca2+ from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.
Muscarinic Receptor Subtypes Mediating Ca2+ Sensitization of Intestinal Smooth Muscle Contraction: Studies with Receptor Knockout Mice
