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 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.
Vajda
Szilvia
S
Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary.
Bartha
Katalin
K
Wilhelm
Imola
I
Krizbai
Istvan A
IA
Adam-Vizi
Vera
V
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
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