Melittin as an epithelial permeability enhancer I: investigation of its mechanism of action in Caco-2 monolayers.
OBJECTIVE
Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2.


METHODS
The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors.


RESULTS
Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4-70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling.


CONCLUSIONS
Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.
Maher
Sam
S
Institute of Technology Tallaght Dublin, Belgard Road, Tallaght, Dublin, Ireland.
Feighery
Linda
L
Brayden
David J
DJ
McClean
Siobhán
S
D000199:Actins / Q000378:metabolism; D034341:Aristolochic Acids / Q000494:pharmacology; D018938:Caco-2 Cells; D015820:Cadherins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007408:Intestinal Absorption / Q000187:drug effects*; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D008555:Melitten / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D010539:Permeability / Q000187:drug effects; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism; D054467:Phospholipases A2; D010750:Phosphoproteins / Q000378:metabolism; D010894:Piroxicam / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011453:Prostaglandins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D062826:Zonula Occludens-1 Protein
D000199:Actins; D034341:Aristolochic Acids; D015820:Cadherins; D016861:Cyclooxygenase Inhibitors; D003911:Dextrans; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D010750:Phosphoproteins; D011453:Prostaglandins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C015219:fluorescein isothiocyanate dextran; D010894:Piroxicam; D008555:Melitten; C000228:aristolochic acid I; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2; D016650:Fluorescein-5-isothiocyanate; D007213:Indomethacin