Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide.
The importance of cell adhesion molecules in maintaining the cellular integrity of the endothelial layer is well recognized, yet their exact participation in regulating the blood-brain barrier (BBB) is poorly understood. Both Ca(2+)-dependent and Ca(2+)-independent cell adhesion molecules are found in endothelial cells. In this study, we used immunofluorescence, ELISA, Western blot and cell adhesion assay to identify a Ca(2+)-dependent cell adhesion molecule, E-cadherin, in bovine brain microvessel endothelial cells (BBMECs). Monoclonal anti-E-cadherin antibody specifically interacted with cultured BBMECs and decorated the cellular junctions with a series of punctate fluorescence spots as seen by indirect immunofluorescence using a confocal microscope. The intensity of these fluorescence spots increased after brief treatment with hIFN-gamma or CPT-cAMP. In the cellular extract of BBMECs, a 120 kDa protein was immunoprecipitated with anti-E-cadherin antibody. BBMECs did not react with anti-N-cadherin antibody, but recognized the FITC-labeled LRAHAVDVNG-NH2, a decapeptide generated from the EC-1 domain of N-cadherin, which decorated the lateral margins of the cells with fluorescence spots. A concentration-dependent binding of this decapeptide was also observed in the flow cytometry assay. BBMECs dissociated with trypsin plus Ca2+ were able to reaggregate only in the presence of Ca2+. However, such cell-cell aggregations of BBMECs were prevented by the presence of either anti-E-cadherin antibody or the decapeptide in the assay medium. These results confirm that BBMECs possess a distinct Ca(2+)-dependent cell adhesion mechanism that can be modulated by the decapeptide. This modulation of cell-cell adhesion in BBMECs by the decapeptide is thought-provoking for creating channels for paracellular drug delivery across the BBB.
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Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66047, USA.
Audus
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KL
Siahaan
T J
TJ
D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002196:Capillaries / Q000166:cytology / Q000187:drug effects; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects*; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007150:Immunohistochemistry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D011233:Precipitin Tests
D015820:Cadherins; D009842:Oligopeptides