Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells.
Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA(2), p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA(2) by Western blot. Capsaicin induced phosphorylation of p38 and cPLA(2). Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA(2) phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA(2). To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.
Ma
Jie
J
Rhode Island Hospital, Gastrointestinal Motor Function Research Laboratory, 55 Claverick Street, Providence, RI 02903, USA.
Harnett
Karen M
KM
Behar
Jose
J
Biancani
Piero
P
Cao
Weibiao
W
D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004947:Esophagus / Q000166:cytology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010972:Platelet Activating Factor / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018689:Sensory System Agents / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D007093:Imidazoles; D010972:Platelet Activating Factor; D011725:Pyridines; D018689:Sensory System Agents; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; D002211:Capsaicin; D002118:Calcium