Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway.
Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr(416) was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 microM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr(416) in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this G(s)-coupled receptor in primary epithelial cells.
Chen
Monica C
MC
CURE/Division of Digestive Diseases, Department of Medicine, School of Medicine, University of California Los Angeles and The Medical and Research Services, Greater Los Angeles Veterans Affairs Health Care System, Los Angeles, California 90073, USA.
Solomon
Travis E
TE
Perez Salazar
Eduardo
E
Kui
Robert
R
Rozengurt
Enrique
E
Soll
Andrew H
AH
D000818:Animals; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004848:Epithelium / Q000502:physiology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D005753:Gastric Mucosa / Q000502:physiology / Q000648:ultrastructure*; D008353:Mannitol / Q000378:metabolism; D010295:Parietal Cells, Gastric / Q000502:physiology / Q000648:ultrastructure; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011799:Quinazolines; D012633:Secretin / Q000494:pharmacology*; D020032:Tyrphostins / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011799:Quinazolines; D020032:Tyrphostins; D012633:Secretin; C101044:RTKI cpd; D019000:Phosphotyrosine; D008353:Mannitol; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases