Lysophosphatidylcholine alters enterocyte monolayer permeability via a protein kinase C/Ca2+ mechanism

Toshio Sawai, Robert Lampman, Yang Hua, Bradley Segura, Robert A. Drongowski, Arnold G. Coran, Caroll M. Harmon

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

The activity of phospholipase A2 (PLA2) is elevated in the intestinal epithelia of patients with inflammatory bowel disease. We recently reported that PLA2 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 Ca2+ mediate TJP in enterocyte monolayer models. To investigate whether the observed change in TJP was mediated via PKC or Ca2+ 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 restistance 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 Ca2+ in the monolayers before and after treatment with L-PC (1 mM) was measured by fluorometry of whole monolayers using the fluorescent calcium indicator fura2. 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 Ca2+ immediately after the addition of L-PC. These results suggest that L-PC alters TJP via a PKC/Ca2+ interaction in our Caco-2 monolayer model.

Original languageEnglish (US)
Pages (from-to)591-594
Number of pages4
JournalPediatric Surgery International
Volume18
Issue number7
DOIs
StatePublished - Oct 2002

Keywords

  • Caco-2 monolayer
  • Protein kinase C
  • Tight-junction permeability

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