Abstract
In single cells, isolated by enzymatic digestion from the circular muscle layer of the lower esophageal sphincter (LES), acute experimental esophagitis (AE) alters signal transduction in response to a maximally effective dose of acetylcholine. In normal LES contraction was inhibited by M3 ≫ M1 or M2antagonists. In AE inhibition by M2 antagonists increased significantly so that contraction was inhibited by M3 > M2 > M1 antagonists. In normal cells permeabilized by saponin, contraction was antagonized by antibodies against Gq/11, by the phosphatidylinositol-specific phospholipase C (PI-PLC) antagonist U 73122, but not by the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609, or by the phospholipase D pathway inhibitor propranolol. In AE contraction was reduced by Gq/11 and Gi3antibodies and by U73122, propranolol and D609. After thapsigargin treatment of normal cells to reduce intracellular Ca++stores, contraction was inhibited by M2 and M3antagonists, by antibodies against Gq/11 and Gi3, by U73122, D609 and propranolol, suggesting that depletion of Ca++ stores reproduces the changes induced by AE. We conclude that in normal LES smooth muscle cells acetylcholine-induced contraction is mediated by M3receptors linked to Gq/11 and PI-PLC, whereas in AE, contraction through this pathway is reduced, perhaps because of reduction in Ca++ stores, and a second pathway is activated by M2 receptors linked to Gi3, PC-PLC and phospholipase D.
Footnotes
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Send reprint requests to: P. Biancani, G.I. Motility Research Lab., SWP5, Rhode Island Hospital & Brown University, 593 Eddy Street, Providence RI 02903.
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↵1 Supported by NIH grant DK 28614, and KOSEF hacsim 961-0704-042–2.
- Abbreviations:
- ACh
- acetylcholine
- AE
- acute experimental esophagitis
- ANOVA
- analysis of the variance
- ATP
- adenosine triphosphate disodium salt
- DAG
- diacylglycerol
- EGTA
- ethylene glycol-bis(β-amino ethyl ether)N,N,N′,N′-tetraacetic acid
- G protein
- guanine nucleotide-binding protein
- Gq/11
- Gi3, Go, Go-i3, Gi1–2 antibody: antibody raised against terminal peptide to respective subfamily of G proteins α subunit
- HEPES
- lN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)
- HPLC
- high-performance liquid chromatography
- 1-IP1
- inositol 1-monophosphate
- 1
- 4-IP2, inositol 1,4-bisphosphate
- 1
- 4,5-IP3, inositol 1,4,5-trisphosphate
- LES
- lower esophageal sphincter
- PKC
- protein kinase C
- PI-PLC
- phosphatidylinositol-specific phospholipase C
- PC-PLC
- phosphatidylcholine-specific phospholipase C
- PLD
- phospholipase D
- PLA2
- phospholipase A2
- pF-HSD
- p-fluoro-hexahydro-sila-difenidol
- Received March 14, 1997.
- Accepted August 1, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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