Lysophosphatidylcholine alters enterocyte monolayer permeability via a protein kinase C/Ca2+ mechanism.
The activity of phospholipase A(2) (PLA(2)) is elevated in the intestinal epithelia of patients with inflammatory bowel disease. We recently reported that PLA(2) mediates the hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine (L-PC) when both are applied to the apical surface of cultures enterocyte monolayers, resulting in increased bacterial translocation (BT) and decreased transepithelial electrical resistance (TEER). However, the mechanism by which the converted L-PC affects tight-junction permeability (TJP) as reflected by decreased TEER is unknown. There are some reports that protein kinase C (PKC) or Ca(2+) mediate TJP in enterocyte monolayer models. To investigate whether the observed change in TJP was mediated via PKC or Ca(2+) in our Caco-2 monolayer model, human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell culture system. The filters were then transferred to an Ussing chamber for precise, real-time resistance measurements. After 30 min equilibration, PC (0.1 or 1 mM) and L-PC (0.01, 0.1 or 1 mM), PMA 200 or 300 nM (phorbol 12-myristate 13-acetate, PCK activator), or staurosporine 12 nM (PKC inhibitor) were added to the apical chamber and TEER was measured every 20 s for 2 h. The concentration of intracellular free Ca(2+) in the monolayers before and after treatment with L-PC (1 mM) was measured by fluorometry of whole monolayers using the fluorescent calcium indicator fura-2. Neither PC at any dose nor the 0.01-mM L-PC dose had an effect on TEER. The 0.1-mM dose of L-PC had its greatest effect (47% +/- 3.5% reduction in TEER vs control) within 6 min following its addition, with TEER recovery to control levels (100%) at 2 h ( P < 0.05). The 1-mM dose of L-PC had its greatest effect (6% +/- 0.5% reduction in TEER vs control) within 3 min after its addition, but the TEER did not recover to control levels after 2 h of incubation ( P < 0.05). The addition of 200 or 300 nM PMA inhibited the observed recovery of TEER by L-PC. Conversely, the addition of 12 nM staurosporine enhanced TEER recovery to control levels. The 1-mM dose of L-PC increased the concentration of intracellular free Ca(2+) immediately after the addition of L-PC. These results suggest that L-PC alters TJP via a PKC/Ca(2+) interaction in our Caco-2 monolayer model.
Sawai
Toshio
T
University of Michigan Medical School, Section of Pediatric Surgery, F3970 Mott Children's Hospital, Ann Arbor, MI 48109-0245, USA.
Lampman
Robert
R
Hua
Yang
Y
Segura
Bradley
B
Drongowski
Robert A
RA
Coran
Arnold G
AG
Harmon
Caroll M
CM
D018938:Caco-2 Cells; D002118:Calcium / Q000502:physiology; D020895:Enterocytes / Q000378:metabolism; D006801:Humans; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D010741:Phospholipases A / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology*; D019108:Tight Junctions / Q000378:metabolism
D008244:Lysophosphatidylcholines; D011493:Protein Kinase C; D010741:Phospholipases A; D002118:Calcium