Mechanical stimulation activates Gαq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells
Minsoo Kim, … , Fievos Christofi, Helen J. Cooke
Minsoo Kim, … , Fievos Christofi, Helen J. Cooke
Published October 1, 2001
Citation Information: J Clin Invest. 2001;108(7):1051-1059. https://doi.org/10.1172/JCI12467.
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Article

Mechanical stimulation activates Gαq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells

Abstract

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-β-S) and a synthetic peptide derived from the COOH terminus of Gαq abolished mechanically evoked 5-HT release, while the NH2-terminal peptide did not. An antisense phosphorothioated oligonucleotide targeted to a unique sequence of Gαq abolished mechanically evoked 5-HT release and reduced Gαq protein levels without affecting the expression of Gα11. 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 Gαq, mobilizes intracellular calcium, and causes 5-HT release.

Authors

Minsoo Kim, Najma H. Javed, Jun-Ge Yu, Fievos Christofi, Helen J. Cooke

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Effects of SLO on LDH activity and 5-HT release. BON cells were permeabi...
Effects of SLO on LDH activity and 5-HT release. BON cells were permeabilized by 20 U/ml SLO for 5 minutes at 37°C in permeabilizing buffer. After permeabilization, SLO was washed out, and BON cells were incubated at 37°C for different time periods from 5 minutes to 115 minutes. (a) Buffers were collected after each time period and the amount of LDH activity was measured. Values represent percentage of total LDH activity (n = 3). (b) After incubations, BON cells were stimulated with/without rotational shaking (80 rpm) and 5-HT release was measured after the stimulation (20 minutes). Values represent concentrations of 5-HT in wells. Linear regression analysis of 5-HT release showed that the slopes were not significantly different (–0.018 ± 0.0050 and –0.032 ± 0.0098; P = 0.21) under static or stimulated conditions (80 rpm; n = 3).