Mechanical stimulation activates Galphaq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells.
5-Hydroxytryptamine (5-HT) released from enterochromaffin cells activates secretory and peristaltic reflexes necessary for lubrication and propulsion of intestinal luminal contents. The aim of this study was to identify mechanosensitive intracellular signaling pathways that regulate 5-HT release. Human carcinoid BON cells displayed 5-HT immunoreactivity associated with granules dispersed throughout the cells or at the borders. Mechanical stimulation by rotational shaking released 5-HT from BON cells or from guinea pig jejunum during neural blockade with tetrodotoxin. In streptolysin O-permeabilized cells, guanosine 5'-O- (2-thiodiphosphate) (GDP-beta-S) and a synthetic peptide derived from the COOH terminus of Galphaq abolished mechanically evoked 5-HT release, while the NH(2)-terminal peptide did not. An antisense phosphorothioated oligonucleotide targeted to a unique sequence of Galphaq abolished mechanically evoked 5-HT release and reduced Galphaq protein levels without affecting the expression of Galpha(11). Depletion and chelation of extracellular calcium did not alter mechanically evoked 5-HT release, whereas depletion of intracellular calcium stores by thapsigargin and chelation of intracellular calcium by 1,2-bis (o-Aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) reduced 5-HT release. Mechanically evoked 5-HT release was inhibited by somatostatin-14 in a concentration-dependent manner. The results suggest that mechanical stimulation of enterochromaffin-derived BON cells directly or indirectly stimulates a G protein-coupled receptor that activates Galphaq, mobilizes intracellular calcium, and causes 5-HT release.
Kim
M
M
Department of Pharmacology, The Ohio State University, College of Medicine, Columbus, Ohio 43210, USA.
Javed
N H
NH
Yu
J G
JG
Christofi
F
F
Cooke
H J
HJ
D001426:Bacterial Proteins; D002021:Buffers; D002118:Calcium; D002276:Carcinoid Tumor; D002614:Chelating Agents; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D012701:Serotonin / Q000378:metabolism*; D015398:Signal Transduction; D013004:Somatostatin / Q000378:metabolism; D013301:Streptolysins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D002021:Buffers; D002614:Chelating Agents; D013301:Streptolysins; C025964:streptolysin O; D012701:Serotonin; D013004:Somatostatin; D007770:L-Lactate Dehydrogenase; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium