Role of chloride ions in lower esophageal sphincter tone and relaxation

Studies were performed in strips of opossum lower esophageal sphincter (LES) muscle in vitro. External Cl(-)-free Krebs solution (0[Cl-]o) inhibited resting tone. Treatment with the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 0.3-100 microM) caused a concentration-dependent relaxation of LES muscle, as did treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1 microM-3 mM), a Cl(-)-HCO3- exchange blocker, and bumetanide (0.3-100 microM), a blocker of the Na(+)-K(+)-2Cl- cotransport. DIDS and bumetanide are also known to cause Cl- channel block. The calculated pD2 and Emax values for DPC, DIDS, and bumetanide were 5.24 +/- 0.28 (n = 5), 3.38 +/- 0.16 (n = 5), and 4.49 +/- 0.23 M (n = 5), and 78.80 +/- 5.38, 74.80 +/- 6.54, and 83.70 +/- 10.20%, respectively. The neuronal Cl- channel activators gamma-aminobutyric acid and glycine had no effect on the resting tone. DPC, DIDS, and bumetanide appear to have acted directly on smooth muscle rather than indirectly through the release of inhibitory neurotransmitters because LES relaxation by these agents was not influenced by tetrodotoxin (10 microM), which blocks action potentials in nerves, or by omega-conotoxin (1 microM), which inhibits the release of neurotransmitters from nerve terminals. LES muscle relaxed by exposure to 0[Cl-]o, DPC, DIDS, and bumetanide contracted with the addition of carbachol (30 microM); muscle so treated was resistant to the inhibitory neurotransmitter-mediated relaxation ordinarily induced by electrical field stimulation (EFS, 0.12-32 Hz). This effect was not nonselective, as the EFS-resistant muscle relaxed fully with isoproterenol (0.1-100 microM). HCO(3-)-free Krebs in the nominal absence of CO2 did not affect the resting tone and its relaxation. The Ca2+ channel blocker nifedipine decreased resting tone but did not antagonize the relaxation of carbachol-contracted muscle induced by either EFS or isoproterenol. These studies suggest that Cl- may play an important role in LES tone and relaxation due to inhibitory neurotransmitter released from intramural nerves.