Angiotensin II‐induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol‐phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris‐phosphate (IP3), intracellular calcium, [Ca2+]i, and protein kinase C (PKC), in angiotensin II (AII)‐induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP3 and a rapid increase in [Ca2+]i in cells loaded with the Ca2+‐responsive fluorescent dye fura‐2. The prompt AII‐induced [Ca2+]i spike was not affected by incubating HCEC in Ca2+‐free medium containing 2 mM EGTA or by pretreating the cultures with the Ca2+ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca2+ from intracellular stores. The AII‐triggered increases in IP3, [Ca2+]i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar1, Val5, Ala8‐AII (SVA‐AII), and by the phospholipase C (PLC) inhibitors, neomycin and U‐73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII‐induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood‐brain barrier permeability in acute hypertension, is likely dependent on PLC‐mediated changes in [Ca2+]i and independent of PKC. © 1996 Wiley‐Liss, Inc.