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Fig. 3. Intracellular ROS contributes to [Ca²⁺]_i-oscillation-regulated VCAM1 gene expression during agonist stimulation. (A) Intracellular-Ca²⁺-store-depleted EC monolayers were exposed to 1 µM histamine stimulation in Ca²⁺-free/EGTA HBS for 60 minutes. This condition, under which histamine does not trigger any [Ca²⁺]_i signal, still upregulates VCAM1 mRNA expression (^*P<0.05 vs intracellular-Ca²⁺-store-depleted control ECs, n=3 for each). The histamine-stimulated VCAM1 mRNA expression in intracellular-Ca²⁺-store-depleted ECs is abolished by NADPH-oxidase inhibition through Rac^–/– expression and this is reversed by an external application of 10 µM H₂O₂. H₂O₂ alone at 10 µM induces VCAM1 mRNA expression in intracellular-Ca²⁺-store-depleted ECs. (B) Rac^–/–-transfected or control-vector-transfected ECs were pre-treated to deplete the intracellular Ca²⁺ store and were then exposed to the conditions generating 0.1, 0.3, 0.5 and 0.7 [Ca²⁺]_i oscillations/minute in the concomitant presence of histamine stimulation for 60 minutes. Total RNA was then isolated for real-time RT-PCR analysis. The bell-shaped regulation of VCAM1 mRNA expression by [Ca²⁺]_i oscillation frequency during histamine stimulation was shifted to the right when H₂O₂ generation was inhibited in Rac^–/–-expressing ECs versus vector-control ECs (n=3 for each).

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