title: Identification of protease-activated receptor-4 (PAR-4) in puromycin-purified brain capillary endothelial cells cultured on Matrigel.
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.
authors.affiliation: Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary.
authors.forename: Szilvia
authors.initials: S
authors.lastname: Vajda
authors.forename: Katalin
authors.initials: K
authors.lastname: Bartha
authors.forename: Imola
authors.initials: I
authors.lastname: Wilhelm
authors.forename: Istvan A
authors.initials: IA
authors.lastname: Krizbai
authors.forename: Vera
authors.initials: V
authors.lastname: Adam-Vizi
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002469:Cell Separation; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000378:metabolism* / Q000648:ultrastructure; D062507:Claudin-5; D003094:Collagen / Q000494:pharmacology*; D004338:Drug Combinations; D042783:Endothelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000378:metabolism / Q000648:ultrastructure; D007797:Laminin / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D010455:Peptides / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000494:pharmacology*; D011691:Puromycin; D051381:Rats; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism*; D013997:Time Factors; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; C107466:CLDN5 protein, human; D062507:Claudin-5; D004338:Drug Combinations; D007797:Laminin; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D011509:Proteoglycans; D018179:Receptors, Thrombin; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; C059485:matrigel; D011691:Puromycin; D003094:Collagen; C112908:protease-activated receptor 4; D002118:Calcium