Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Abstract

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.

Tachykinin Receptor Expression and Function in Human Esophageal Smooth Muscle

“…Tissue collection was carried out in accordance with the guidelines of The University of Western Ontario Research Ethics Board for the Review of Health Sciences Research Involving Human Subjects. Tissues were obtained from patients undergoing esophageal resection as described previously (Preiksaitis and Diamant, 1997;Sims et al, 1997;Kovac et al, 2005). Samples were removed from diseasefree regions of the distal third, placed in ice-cold, oxygenated Krebs bicarbonate solution (see below), and transported to the laboratory.…”

“…Portions of muscle were frozen on dry ice (Ϫ70°C) for RNA extraction or further dissected for acute studies. Freshly dispersed SMCs were studied within 6 h or maintained in primary culture as described previously (Sims et al, 1997;Wang et al, 2000;Kovac et al, 2005). In total, muscle was studied from 55 specimens.…”

“…Muscle strips were mounted in tissue baths containing 10 ml of Krebs bicarbonate solution continuously bubbled with 5% CO 2 -95% O 2 at 37°C as described previously (Sims et al, 1997;Wade et al, 1999;Kovac et al, 2005).…”

“…Cells were loaded by incubation with fura-2-acetoxymethyl ester (0.2 M) or fluo-4-acetoxymethyl ester (5 M) at room temperature (21-24°C) for 40 min, as described previously (Kovac et al, 2005). Cells loaded with fluo-4 were illuminated with 488 nm of light from an argon ion laser and emissions detected at 510 to 560 nm using a Photometrics Cascade camera (Photometrics, Tucson, AZ) controlled by PTI ImageMaster software (Photon Technology Int.…”

“…Dispersed cells were allowed to settle and adhere to the bottom of a perfusion chamber mounted on the stage of a Nikon inverted microscope (Nikon, Tokyo, Japan) and perfused with Na ϩ -HEPES bathing solution, as described previously (Kovac et al, 2005). Dispersed cells were studied at room temperature (21-24°C).…”