Abstract
An improved method is described for culturing primary rat brain capillary endothelial cells (RBCEC) on glass, covered by Matrigel. The procedure using Matrigel yields spindle-shaped endothelial cells exhibiting close cell-cell appositions seen on electron microscopic sections. These cells permanently express tight junction proteins ZO-1, claudin-5 and the adherent junction protein beta-catenin, as revealed by immunofluorescence. Furthermore, glass coverslips covered with Matrigel provide a stable and low-background fluorescent base for microfluorimetric calcium measurements. By this method, hereby we show that the PAR-4 agonist peptide induces transient [Ca2+]i changes with different kinetics compared to that due to activation of the PAR-1 receptor. This indicates that RBCE cells grown on Matrigel express PAR-4 receptors.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biocompatible Materials / pharmacology
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Calcium / analysis
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Calcium / metabolism
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Calcium Signaling / drug effects
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Calcium Signaling / physiology
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Cell Culture Techniques
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Cell Differentiation / drug effects
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Cell Differentiation / physiology
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Cell Separation
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Cells, Cultured
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Cerebral Arteries / metabolism*
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Cerebral Arteries / ultrastructure
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Claudin-5
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Collagen / pharmacology*
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Drug Combinations
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Endothelial Cells / metabolism*
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Endothelial Cells / ultrastructure
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Immunohistochemistry
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Intercellular Junctions / metabolism
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Intercellular Junctions / ultrastructure
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Laminin / pharmacology*
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Membrane Proteins / metabolism
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Microscopy, Electron, Transmission
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Peptides / pharmacology
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Phosphoproteins / metabolism
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Protein Synthesis Inhibitors / pharmacology
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Proteoglycans / pharmacology*
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Puromycin
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Rats
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Receptors, Thrombin / agonists
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Receptors, Thrombin / metabolism*
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Time Factors
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Zonula Occludens-1 Protein
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beta Catenin / metabolism
Substances
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Biocompatible Materials
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CLDN5 protein, human
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Claudin-5
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Drug Combinations
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Laminin
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Membrane Proteins
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Peptides
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Phosphoproteins
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Protein Synthesis Inhibitors
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Proteoglycans
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Receptors, Thrombin
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TJP1 protein, human
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Tjp1 protein, rat
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Zonula Occludens-1 Protein
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beta Catenin
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matrigel
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Puromycin
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Collagen
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protease-activated receptor 4
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Calcium