Identification of protease-activated receptor-4 (PAR-4) in puromycin-purified brain capillary endothelial cells cultured on Matrigel

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 β-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.