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Fig. 6. Agonist stimulation increases the efficiency of [Ca²⁺]_i-oscillation-frequency-regulated NF-B transcriptional activation. (A) EC monolayers were exposed to conditions that generated [Ca²⁺]_i oscillations with the same amplitude of 0.9 µM and four different oscillation frequencies of 0.1, 0.3, 0.5 or 0.7 oscillations/minute, in the presence or absence of 1 µM histamine stimulation. Then, cell lysates were prepared for subsequent ELISA-based assay for determination of endogenous NF-B transcriptional activity. Histamine stimulation shifts the frequency–NF-B-transcriptional-activity curve to the left and decreases the optimal [Ca²⁺]_i oscillation frequency from 0.45 oscillations/minute in the absence of histamine to 0.3 oscillations/minute in the presence of histamine stimulation (n=3 for each). (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 or 0.7 oscillations/minute in the concomitant presence of histamine stimulation for 60 minutes. Cell lysates were then isolated for subsequent ELISA-based assay for determination of endogenous NF-B transcriptional activity. The bell-shaped regulation curve of NF-B transcriptional activity versus [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). (C) Intracellular-Ca²⁺-store-depleted ECs were exposed to the conditions generating 0.1, 0.3, 0.5 and 0.7 oscillations/minute in the presence of 10 µM H₂O₂, or in the concomitant presence of 10 µM H₂O₂ and 1 µM histamine stimulation, in Rac^–/–-expressing ECs for 60 minutes. Then, cell lysates were isolated for subsequent ELISA-based assay for determination of endogenous NF-B transcriptional activity. An external application of 10 µM H₂O₂ reverses the altered bell-shaped graph of histamine-stimulated NF-B transcriptional activity versus [Ca²⁺]_i oscillation frequency that is induced by Rac^–/– expression (see B) to that of vector controls. In the absence of histamine stimulation, the external application of 10 µM H₂O₂ cooperates with [Ca²⁺]_i oscillation frequency to optimize NF-B transcriptional activation.

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