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-phosp hate (IP3), intracellular calcium, [Ca2+](i), and protein kinase C (PK C), in angiotensin II (AII)-induced fluid phase endocytosis was invest igated in human brain capillary and microvascular endothelial cells (H CEC). AII (0.01-10 mu M) stimulated the uptake of Lucifer yellow CH, a n inert dye used as a marker for fluid phase endocytosis, in HCEC by 5 0-230%. AII also triggered a fast accumulation of IP3 and a rapid incr ease in [Ca2+](i) in cells loaded with the Ca2+-responsive fluorescent dye fura-2. The prompt AII-induced [Ca2+](i) spike was not affected b y incubating HCEC in Ca2+-free medium containing 2 mM EGTA or by pretr eating the cultures with the Ca2+ channel blockers, methoxyverapamil ( D600; 50 mu M), nickel (1 mM), or lanthanum (1 mM), suggesting that th e activation of AII receptors on HCEC triggers the release of Ca2+ fro m intracellular stores. The AII-triggered increases in IP3, [Ca2+](i), and Lucifer yellow uptake were inhibited by the nonselective AII rece ptor antagonist, Sar(1), Val(5), Ala(8)-AII (SVA-AII), and by the phos pholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the p rotein kinase C (PKC) inhibitors, staurosporine and calphostin C, fail ed 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, i s likely dependent on PLC-mediated changes in [Ca2+](i) and independen t of PKC. (C) 1996 Wiley-Liss, Inc.